Carbohydrate sulfotransferase 14 gene deletion induces dermatan sulfate deficiency and affects collagen structure and bowel contraction

Fumiko Ono; Yuki Takahashi; Shin Shimada; Shuji Mizumoto; Shinji Miyata; Yuko Nitahara-Kasahara; Shuhei Yamada; Takashi Okada; Tomoki Kosho; Takahiro Yoshizawa

doi:10.1371/journal.pone.0320943

Abstract

Dermatan sulfate (DS) is a type of glycosaminoglycan present in the extracellular matrix, and which is related to tissue strength, structure, and healing. Dermatan 4-O-sulfotransferase 1 (D4ST1) is an enzyme that catalyzes the transfer of a sulfate group to the N-acetylgalactosamine residue of dermatan, resulting in mature DS. Biallelic loss-of-function variants in the carbohydrate sulfotransferase 14 (CHST14) gene encoding D4ST1, induce defective DS biosynthesis. DS deficiency causes severe connective tissue fragility and deformities in humans (musculocontractural Ehlers–Danlos Syndrome [mcEDS]) and mice (Chst14 gene knockout [Chst14^-/-] mice). Many patients with mcEDS experience gastrointestinal symptoms such as constipation, diverticula, diverticulitis, and perforation. However, pathogenesis of these symptoms has not been systematically investigated. Therefore, we sought to determine the effects of DS deficiency on the colon using Chst14^-/- mice. We found that collagen fibrils were abnormally arranged in the submucosa of the colon. The mice also exhibited accelerated colonic contraction. Unexpectedly, no significant aggravation of dextran sulfate sodium-induced colitis was observed in Chst14^-/- mice compared with wild-type mice. These findings suggest a physiological role of DS in the colon and may shed light on the potential mechanisms underlying the gastrointestinal symptoms of mcEDS.

Citation: Ono F, Takahashi Y, Shimada S, Mizumoto S, Miyata S, Nitahara-Kasahara Y, et al. (2025) Carbohydrate sulfotransferase 14 gene deletion induces dermatan sulfate deficiency and affects collagen structure and bowel contraction. PLoS One 20(5): e0320943. https://doi.org/10.1371/journal.pone.0320943

Editor: Nikos K. Karamanos, University of Patras, GREECE

Received: December 10, 2024; Accepted: February 26, 2025; Published: May 6, 2025

Copyright: © 2025 Ono et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: JSPS KAKENHI Grant Numbers JP23K07780 (to T.Y.) and 23K06142 (to S.M.). Grant-in-Aid by the Nagano Society for the Promotion of Science (to T.Y.). Grant-in-Aid for Scientific Research (A) from the Japan Society for the Promotion of Science, Grant Number 24H00646, and AMED under Grant Number, JP24bm1523007 (to T.O.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: Y.T. and T.K. are members of an endowed chair named “Division of Clinical Sequencing, Shinshu University School of Medicine” sponsored by BML Inc. and Life Technologies Japan Ltd. of Thermo Fisher Scientific Inc.

Introduction

Dermatan sulfate (DS) is present in the extracellular matrix of various tissues such as the skin, tendon, and colon [1]. DS is a type of glycosaminoglycan (GAG), which is found in chondroitin sulfate (CS)/DS long polysaccharide chains composed of repeating disaccharide units of glucuronic acid (GlcA) or iduronic acid (IdoA) and N-acetylgalactosamine (GalNAc). These polysaccharide chains bind to serine residues of core proteins, forming proteoglycans (PGs) [2–4]. CS/DS-PGs interact with diverse molecules including matrix molecules, growth factors, protease inhibitors, cytokines, and chemokines, and are involved in various biological processes such as anticoagulation, infection, healing, and extracellular matrix construction [3]. DS is distinguished from CS by epimerized IdoA at C-5 of GlcA [3]. Dermatan sulfate epimerase (DSE) epimerizes GlcA residues to IdoA residues by C-5 inversion at the polymer level of the substrate, (-GlcAβ-1,3-GalNAcβ-1,4-)_n. Shortly thereafter, C4 of GalNAc (in the IdoA-GalNAc sequence) is sulfated by dermatan-4-sulfotransferase-1 (D4ST1) [5–7]. This sulfation prevents reverse epimerization from IdoA to GlcA, thereby completing DS biosynthesis [7,8].

DSE and D4ST1 are encoded by DSE and carbohydrate sulfotransferase 14 (CHST14), respectively. Biallelic loss-of-function variants in CHST14 and DSE result in defective DS biosynthesis, leading to musculocontractural Ehlers–Danlos Syndrome (mcEDS) [9–12]. DS is undetectable in skin fibroblasts or urine samples from patients with mcEDS-CHST14 [10,13], and significantly reduced in skin fibroblasts or undetectable in urine samples from patients with mcEDS-DSE [11,14]. Patients with mcEDS exhibit characteristic clinical manifestations, including multiple malformations and progressive connective tissue fragility-related complications [12,15,16]. An international collaborative study on mcEDS-CHST14 reported gastrointestinal symptoms as common manifestations, including constipation (85%) and diverticula (35%) [16]. Several patients developed fatal colonic perforations [16–18].

Chst14 gene knockout (Chst14^-/-) mice and mice generated using CRISPR/Cas9-mediated genomic editing for Chst14 lack DS [19,20]. Phenotypic features of these mice include postnatal generalized growth disturbance [21], skin fragility with decreased skin tensile strength [21,22], thoracic kyphosis [20], embryonic lethality thought to be associated with placental vascular abnormalities [21,23], and tooth and tail abnormalities [21]. These skin and spine features reflect those observed in patients with mcEDS-CHST14 [24]. However, the functions of DS and CS/DS-PGs in the intestinal tract have not been investigated.

Therefore, we used Chst14^-/- mice to determine the effects of DS deficiency on the gastrointestinal tract, particularly the colon. We found that Chst14^-/- mice had abnormally arranged collagen fibrils in the submucosa of the colon. They exhibited accelerated intestinal contraction without constipation or diverticula. Unexpectedly, no significant aggravation of dextran sulfate sodium (DSS)-induced colitis was observed in Chst14^-/- mice compared with wild-type (Chst14^+/+) mice. These results suggest a role for DS in the colon and also as the pathological basis of gastrointestinal symptoms in mcEDS.

Materials and methods

Animal studies

The mouse strain, B6;129S5-Chst14^tm1Lex/Mmucd (identification number 031629-UCD), was obtained from the Mutant Mouse Regional Resource Center (MMRRC; UC Davis, Sacramento, CA, USA; https://www.mmrrc.org/) [25], an NCRR/NIH-funded strain repository, and was donated to the MMRRC by Lexicon Genetics Inc and backcrossed on a BALB/cAJc1(BALB/cA) inbred strain (CLEA Japan Inc., Shizuoka, Japan) [26]. Mice were housed at a constant temperature of 23 ± 3 °C, with a relative humidity of 45%–70% and a 12-hour light/dark cycle. Animals had free access to tap water and standard mouse chow (Funabashi Farm, Chiba, Japan). In this study, male mice were used to ensure consistency in the experimental conditions. This approach minimizes gender-related variations in body weight and colon length, allowing for a more direct evaluation of the effects of the experimental conditions. In the present study, no invasive treatment was performed on the live mice. Animal welfare was closely monitored throughout the experiments, and no severe pain was observed in the mice. The mice were euthanized by cervical dislocation before sample collection. All experimental procedures were performed in accordance with the Regulations for Animal Experimentation of Shinshu University. The animal protocol was reviewed by the Committee for Animal Experiments of Shinshu University based on national regulations and guidelines, and approved by the president of Shinshu University (Approval number: 022010).

Genotyping

The PCR method was reported in our previous studies [23,26]. Ear specimens were collected from 3-week-old mice and DNA was extracted using Mighty Prep reagent (Takara Bio Inc., Shiga, Japan). Primer sequences for wild-type genotyping designed to exon 1 of the Chst14 gene were: 5′-GGACCACCGCAGTGACTTG-3′ and 5′-ACAGGCATCCAATGCTCATTC-3′. Primer sequences for the neomycin resistance gene in knockout PCR were: 5′-TGGCTCTCCTCAAGCGTATT-3′ and 5′-GTTTTCCCAGTCACGACGTT-3′. PCR was performed using TaKaRa Taq™ HS Perfect Mix (Takara Bio Inc.). The PCR conditions were 94 °C for 1 minute followed by 30 cycles at 94 °C for 5 seconds and then 65 °C for 15 seconds. PCR products were visualized using agarose gel electrophoresis.

Analysis of food intake, body weight, and fecal volume

To avoid coprophagy, 7-week-old male mice were housed individually in wire mesh cages and allowed to acclimatize for 4 days. Food intake, body weight, and fecal volume were measured at fixed times. The defecation volume was measured using a precision balance (Shimadzu Corporation, Kyoto, Japan).

Bead expulsion time

Ten to thirteen-week-old male mice were anesthetized with 1.5% isoflurane (Pfizer Inc., Manhattan, NY, USA) using an inhalation anesthesia apparatus (MK-AT210D; Muromachi Kikai Co., Ltd., Kyoto, Japan). A 5-mm bead was inserted into the anus and the time to expulsion was measured.

Intestinal transit time

Ten to eleven-week-old male mice were orally administered 200 µ L of 6% carmine red and 0.5% methylcellulose (Fujifilm Wako Pure Chemical Co., Osaka, Japan) in phosphate-buffered saline (PBS). The cages were inspected every 10 minutes after oral administration, and time of appearance of the first red fecal pellet was recorded [27].

Dextran sulfate sodium-induced colitis model

Colon injury was induced by 2.0% (weight/volume) DSS (molecular weight 36,000–50,000 g/mol; MP Biomedicals, Santa Ana, CA, USA) in drinking water [28]. Eight-week-old male Chst14^+/+ and Chst14^-/- mice were randomly assigned to three groups: Control group, 2.0% DSS (day 8) group, and 2.0% DSS (day 15) group. The 2.0% DSS groups were given 2.0% DSS solution in drinking water ad libitum for 7 days, while the Control group received sterilized water under the same conditions. Dissections were performed on day 8 for the Control group and 2.0% DSS (day 8) group, and on day 15 for the 2.0% DSS (day 15) group. Body weight, fecal characteristics, and water intake were monitored daily at 17:00 ± 2 hours. The disease activity index (DAI) score, which is a composite measure of weight loss, stool bleeding, and stool consistency, was determined as previously reported [29–31]. Body weight loss: 0 (no loss), 1 (1%–5% loss), 2 (5%–10% loss), 3 (10%–20% loss), and 4 (> 20% loss). Stool consistency: 0 (normal), 2 (loose stool), and 4 (diarrhea, gross bleeding). Hematoxylin and eosin (H&E) scoring was used for histological determination of colon injury [32,33]. Inflammation: 0 (no inflammation), 1 (mild), 2 (moderate), and 3 (severe). Inflammatory cell infiltration: 0 (none), 1 (mucosal layer), 2 (submucosal layer), and 3 (all muscle layers). Injury to crypts: 0 (1/3), 1 (2/3), 3 (surface only), and 4 (loss of mucosal layer); and range: 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%).

Sample collection

Mice were euthanized and their colons were collected. Photographs of the tissue were obtained. Colons were then washed in PBS and fixed with 4% paraformaldehyde, or stored at −80 °C. For experiments with nifedipine (Fujifilm Wako Pure Chemical Co.), colons were immersed in PBS containing 4 µ M nifedipine for 10 minutes.

Strength and extensibility test

Colons of male mice aged 8–10 weeks were cut along the longitudinal axis into cylindrical strips 3 mm wide. A 10-g weight was loaded and the length of each extended colon sample was measured. Colon strength (force required for rupture) was measured using a dynamometer (Muromachi Kikai Co., Ltd.).

Pathological analysis

Samples were fixed in 4% paraformaldehyde for 48 hours at 4 °C. After dehydration in an ethanol and xylol series, whole colon samples were embedded in paraffin and cut into 5 µm thick sections. Sections were deparaffinized and then subjected to hydrophilic treatment by stepwise ethanol washes. This was followed by deionized water and then used for H&E, Sirius red, and Alcian blue staining. Thirty random locations were selected from each sample stained with H&E to measure the mucosal thickness, mucosal muscle, submucosa, and muscle layers using ImageJ software [34]. H&E staining and H&E scores were used to determine the extent of tissue damage. To observe collagen components in the colon, Sirius red staining was performed using Weigert’s iron hematoxylin and Von Gieson’s solution, according to the manufacturer’s instructions (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). Alcian blue staining using 3% acetic acid (Fujifilm Wako Pure Chemical Corp.,), Alcian blue solution, and Kernechtrot solution (Muto Pure Chemicals Co., Ltd.) was performed to stain mucins and GAGs for assessment of damage to the glandular ducts of the mucosal layer.

Immunofluorostaining of lymphocyte antigen-6 family member G (Ly-6G)

Immunofluorostaining of Ly-6G was performed on 6 µm thick paraffin sections. After deparaffinization and hydrophilic treatment, antigen retrieval was performed by autoclaving with HISTOFINE Depara Antigen Activation Solution, pH 6 (Nichirei, Tokyo, Japan). After blocking with 2% bovine serum albumin, sections were incubated overnight at 4 °C with a 1:50 dilution of Alexa Fluor 488-conjugated Ly-6G antibody (Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were then stained with a 1:1000 dilution of 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Waltham, MA, USA). Stained sections were observed using a fluorescence microscope (Olympus, Tokyo, Japan). Ly-6G-positive cells in all sections were counted three times and averaged.

Transmission electron microscopy (TEM)

TEM was used to observe the condition of collagen fibrils. Colon tissue was sectioned using a scalpel and fixed with 2.5% glutaraldehyde and 4% osmium tetroxide. It was then embedded in epoxy resin, cut into ultrathin sections, and stained with uranyl acetate and lead citrate. Carbon shadowing was applied, and sections were observed by TEM (JEM-1400; JEOL, Tokyo, Japan). Three samples from each group of Chst14^+/+ and Chst14^-/- mice were used to measure collagen fibril diameters using ImageJ software [34].

Scanning electron microscopy (SEM)

Fresh colon samples were fixed with 2.5% glutaraldehyde for 2 days at 4 °C. They were then treated with 8% NaOH at room temperature for 7–10 days, followed by washing with distilled water for 3–7 days. Samples were treated with 1% tannic acid for 2 hours and washed with distilled water. Next, samples were postfixed in 1.0% osmium tetroxide in 0.1 M phosphate buffer for 1 hour at room temperature and washed with distilled water. Samples were then dried using the t-butyl alcohol freeze-drying method, mounted on metal stubs, coated with platinum, and observed by SEM (JSM-7600F; JEOL) with an acceleration voltage of 5 kV.

Western blot analysis

Colon tissues from 10-week-old male mice were homogenized in PBS containing 1% Triton X-100 and a protease inhibitor cocktail (Merck, Darmstadt, Germany), followed by incubation on ice for 30 minutes. The homogenates were centrifuged at 20,000 × g for 30 minutes at 4 °C, and protein concentrations in the supernatants (colon lysates) were measured using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Colon lysates (100 µ g protein) were treated with 5 milliunits of chondroitinase ABC (Merck, Darmstadt, Germany) or chondroitinase B (R&D Systems, Minneapolis, MN, USA) at 37 °C for 4 hours. Both digested and undigested lysates were denatured with 2% sodium dodecyl sulfate and 5% mercaptoethanol at 60 °C for 20 minutes. Proteins were separated by 7.5% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 2% skim milk in PBS containing 0.1% Tween-20 and then incubated overnight at 4 °C with primary antibodies: anti-mouse decorin (DCN) antibody (goat IgG, AF1060, 1:2000, R&D Systems, Minneapolis, MN, USA) or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (mouse IgG, clone 5A12, 1:10,000, Fujifilm Wako Pure Chemical Co., Osaka, Japan). After washing, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:4000) for 1 hour at room temperature. Protein signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Merck, Darmstadt, Germany) and imaged using a LuminoGraph I imaging system (ATTO, Tokyo, Japan).

Quantitative analysis of DS and CS disaccharides

Colons were washed, homogenized, and sonicated. Extraction and purification of GAG fractions were performed as described previously [35]. Samples were treated individually with chondroitinase B (EC4.2.2.19) (R&D Systems, Minneapolis, MN, USA) or a mixture of chondroitinase ABC and AC-II (EC4.2.2.5) (Seikagaku Corp., Tokyo, Japan), respectively. The disaccharide composition of DS or CS/DS moieties on CS/DS chains was analyzed. Digests were labeled with the fluorophore, 2-aminobenzaimide (2AB), and samples were analyzed for each disaccharide by anion-exchange high-performance liquid chromatography (HPLC) on a PA-G column (YMC Co., Kyoto, Japan), as described previously [35]. Disaccharide standards were also labeled with 2AB and analyzed by HPLC. The standards used were: ΔHexUA-GalNAc, ΔHexUA-GalNAc(6S), ΔHexUA-GalNAc(4S), ΔHexUA(2S)-GalNAc(6S), ΔHexUA(2S)-GalNAc(4S), ΔHexUA-GalNAc(4S,6S), and ΔHexUA(2S)-GalNAc(4S,6S). Unsaturated DS and CS/DS disaccharides observed in digests were identified by comparison with elution positions of 2AB-labeled disaccharide standards and quantitated based on peak area relative to standard unsaturated disaccharides.

Real-time quantitative reverse transcription PCR

Frozen colons were homogenized in TRI reagent (Molecular Research Center, Cincinnati, OH, USA) using a Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, CT, USA) to extract total RNA. Homogenates were treated with DNase and RNA then purified with a RNA Clean and Concentrator Kit (Zymo Research, Irvine, CA, USA). RNA was subjected to reverse transcription to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Quantitative PCR was performed using a QuantStudio 3 Real-Time PCR system (Applied Biosystems) with Thunderbird Next SYBR qPCR Mix (Toyobo, Osaka, Japan). Values were normalized to 18S ribosomal RNA levels. The primer sequences are listed in S1 Table.

Statistical analysis

Data are reported as mean ± standard error of the mean (SEM). Statistical comparisons between groups were performed using GraphPad Prism software Ver 10.2.3 (GraphPad Software, San Diego, CA, USA). Differences between two groups were assessed using unpaired two-tailed Student’s t-tests. Data from groups of three or more were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer post hoc test for multiple comparisons. In all analyses, P-values < 0.05 were considered statistically significant.

Results

DS is absent from the colon of Chst14^-/- mice

The DS or CS/DS moiety on CS/DS chains from colon samples were digested into disaccharides with chondroitinase ABC and AC-Ⅱ or chondroitinase B, respectively. HPLC profiles were obtained (Fig 1A–D). Regarding CS/DS disaccharide levels, ΔHexUA-GalNAc, ΔHexUA(2S)-GalNAc(6S), and ΔHexUA(2S)-GalNAc(4S,6S) were not detected (∆HexUA[2S, 4S, and 6S] represent 4,5-unsaturated hexuronic acid, 2-O-, 4-O-, and 6-O-sulfate, respectively). DS disaccharides were only detected in Chst14^+/+ mice and not Chst14^-/- mice. ΔHexUA-GalNAc(6S) and ΔHexUA-GalNAc(4S,6S) are component solely of CS disaccharides. Only ΔHexUA-GalNAc(6S) was significantly increased in Chst14^-/- mice. CS disaccharide levels of ΔHexUA-GalNAc(4S), which is component of CS/DS disaccharides, were significantly higher in Chst14^-/- mice than that of Chst14^+/+ mice. Mean but while, CS/DS disaccharide levels were significantly lower in Chst14^-/- mice than that of Chst14^+/+ mice. ΔHexUA(2S)-GalNAc(4S) was mostly composed of DS disaccharides. Total CS/DS disaccharides were significantly lower in Chst14^-/- mice than Chst14^+/+ mice (Fig 1E). Western blot was performed to analyze GAG modifications on DCN, a major CS/DS-PG, in the colon of Chst14^+/+ and Chst14^-/- mice. In undigested colon lysates, DCN from both Chst14^+/+ and Chst14^-/- mice showed high molecular weight signals indicative of GAG modification. When digested with chondroitinase ABC, which degrades both DS and CS, and chondroitinase B, which specifically degrades DS, DCN from Chst14^+/+ mice shifted similarly to a lower molecular weight corresponding to the position of the core protein. This finding suggests that DCN in the colon of Chst14^+/+ mice is predominantly modified by DS. In contrast, DCN from the colon of Chst14^-/- mice was resistant to chondroitinase B but was completely digested by chondroitinase ABC. These results indicate that in the absence of Chst14, DS on DCN in the colon is significantly reduced and replaced with CS (Fig 1F). Chst14 gene expression was not detected in Chst14^-/- mice. Expression of other genes related to CS/DS synthesis (Chst3, Chst11, Chst12, Chst15, uronyl 2-sulfotransferase [Ust], and Dse) were not significantly different between the two groups of mice (Fig 1G).

Download:

Fig 1. HPLC profiles of DS and CS/DS moieties and expression levels of CS/DS synthesis genes.

(A) HPLC profiles of CS/DS digests (A, C) and DS (B, D) moieties in CS/DS chains prepared from colons of Chst14^+/+ mice (A, B) and Chst14^-/- mice (C, D). Colons were digested into disaccharides for analysis of CS/DS or DS moieties using chondroitinase ABC and AC-II (A, C) or chondroitinase B (B, D), respectively. Each digest was labeled with 2AB, and labeled CS/DS disaccharides were separated by anion-exchange HPLC on amine-bonded silica PA-G columns using a linear gradient of NaH₂PO₄, as indicated by the dashed line. The amounts of disaccharides in each sample were calculated based on peak area relative to standard unsaturated disaccharides. Elution positions of standard 2AB-labeled CS/DS disaccharides are indicated by numbered arrows: 1, ∆ HexUA-GalNAc; 2, ∆ HexUA-GalNAc(6S); 3, ∆ HexUA-GalNAc(4S); 4, ∆ HexUA(2S)-GalNAc(6S); 5, ∆ HexUA(2S)-GalNAc(4S); 6, ∆ HexUA-GalNAc(4S,6S); and 7, ∆ HexUA(2S)-GalNAc(4S,6S), where ∆HexUA, 2S, 4S, and 6S represent 4,5-unsaturated hexuronic acid, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively. An asterisk indicates ∆HexUA-N-acetylglucosamine derived from hyaluronan. (E) Disaccharide composition of CS/DS, CS, and DS in Chst14^-/- and Chst14^+/+ mice. The disaccharide contents of CS/DS and DS were calculated from the peak areas of the HPLC profiles the CS disaccharide content was obtained by subtracting DS from CS/DS. Each value is the mean ± SEM. Chst14^+/+ and Chst14^-/- mice (n = 3 per group). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, t-test. N.D.: not detected (< 0.1 pmol/mg protein). (F) Upper panel: Western blot analysis of DCN in colon lysates from Chst14^+/+ and Chst14^-/- mice after digestion with chondroitinase B or chondroitinase ABC. Lower panel: Western blot analysis of GAPDH used as a loading control. (G) Relative mRNA levels (mean ± SEM) of Chst3, Chst11, Chst12, Chst14, Chst15, Ust, and Dse in colon samples from Chst14^+/+ and Chst14^-/- mice (n = 3 per group). ^****P < 0.0001, t-test. DS, dermatan sulfate; CS, chondroitin sulfate; HPLC, high-performance liquid chromatography; Chst3, carbohydrate sulfotransferase 3; Chst11, carbohydrate sulfotransferase 11; Chst12, carbohydrate sulfotransferase 12; Chst14, carbohydrate sulfotransferase 14; Chst15, carbohydrate sulfotransferase 15; Ust, uronyl-2-sulfotransferase; Dse, dermatan sulfate epimerase; Chst14^+/+ mice, + / + ; Chst14^-/- mice, -/-.

https://doi.org/10.1371/journal.pone.0320943.g001

Chst14^-/- mice have shorter colons with increased contractility

The colons of 10-week-old Chst14^-/- mice were shorter compared with Chst14^+/+ mice. However, no anatomical abnormalities or diverticula were observed (Fig 2A, C). Chst14^-/- mice weighed significantly less than Chst14^+/+ mice (Fig 2B). To account for weight-related biases, we compared the colon length of 10-week-old Chst14^-/- mice with 6-week-old Chst14^+/+ mice matched for body weight (Fig 2D). Even after this adjustment, the colons of Chst14^-/- mice were still significantly shorter (Fig 2E). Nifedipine, a calcium channel blocker, was used to investigate the effect of intestinal contraction on colon shortening. Nifedipine suppressed intestinal contractions and eliminated the difference in colon length between Chst14^+/+ and Chst14^-/- mice (Fig 2A, C).

Download:

Fig 2. Appearance and physiological function of the colon.

(A) Typical appearance of control colon and colon treated with nifedipine. (B) Body weight (mean ± SEM). Control 10-week-old Chst14^+/+ and Chst14^-/- mice (n = 7 per group). ^**P < 0.01, t-test. (C) Comparison of colon length between control and nifedipine groups (mean ± SEM). Control Chst14^+/+ mice, n = 7; and Chst14^-/- mice, n = 7; and Nifedipine Chst14^+/+ mice, n = 8; and Chst14^-/- mice, n = 7. Data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test. ^*P < 0.05, ^**P < 0.01 compared with Chst14^+/+ control group. ^####P < 0.0001 compared with control Chst14^-/- group. (D) Body weight comparison with 6-week-old Chst14^+/+ mice (mean ± SEM). 10-week-old Chst14^+/+ and Chst14^-/- mice, n = 7 per group; and 6-week-old Chst14^+/+ mice, n = 8. Data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test. ^**P < 0.01 compared with 10-week-old Chst14^+/+ group. (E) Comparison of colon length in weight-matched groups. Colon length of 10-week-old Chst14^+/+ and Chst14^-/- mice, n = 7 per group; and 6-week-old Chst14^+/+ mice, n = 8. Mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test. ^**P < 0.01 compared with 10-week-old Chst14^+/+ group. ^####P < 0.0001 compared with 10-week-old Chst14^-/- group. (F) Amount of defecation per day. Chst14^+/+ and Chst14^-/- mice, n = 8 per group. (G) Daily food intake. Chst14^+/+ and Chst14^-/- mice, n = 8 per group. (H) Gastrointestinal transit time. Chst14^+/+ mice, n = 9; and Chst14^-/- mice, n = 8. (I) Bead ejection time. Chst14^+/+ mice, n = 9; and Chst14^-/- mice, n = 6. (J) Rate of colon extension when loaded with a 10 g weight. Chst14^+/+ mice, n = 12; and Chst14^-/- mice, n = 6. (K) Load required for colon rupture. Chst14 ^+/+ mice, n = 12; and Chst14^-/- mice, n = 6. (F–K) Analyzed using t-test. Mean ± SEM. Chst14^+/+ mice, + / + ; Chst14^-/- mice, -/-.

https://doi.org/10.1371/journal.pone.0320943.g002

Physiological colon function does not differ between Chst14^+/+ and Chst14^-/- mice

There were no differences in the amount of daily defecation or food consumption between Chst14^+/+ and Chst14^-/- mice (Fig 2F, G). There was also no difference in gastrointestinal transit time (Fig 2H). Additionally, there was no difference in the rectoanal reflex, determined by inserting a bead into the rectum and recording the time of expulsion (Fig 2I). The rate of colon extension and load required for colon rupture showed no significant differences between Chst14^+/+ and Chst14^-/- mice (Fig 2J, K).

Collagen fibril deformation is induced by DS deficiency in the colon of Chst14^-/- mice

H&E staining revealed no histological differences in distal colon tissue between Chst14^+/+ and Chst14^-/- mice (Fig 3A). There were no differences in the thickness of each tissue layer (Fig 3B). Sirius red is used to stain collagen fibers, which are mainly localized in the submucosa (Fig 3A). These submucosal collagen fibrils were observed using TEM, and found to be sparse and disorganized in Chst14^-/- mice (Fig 3A). Using SEM, Chst14^-/- mice had a lower density of collagen fibrils than Chst14^+/+ mice (Fig 3A). Collagen fibrils had significantly smaller diameters in Chst14^-/- mice than Chst14^+/+ mice (Fig 3C). There were no differences in gene expression of collagen 1 alpha-1 (Col1a1) and collagen 3 alpha-1 (Col3a1), components of collagen in the colon (Fig 3D). There were also no differences in gene expression of Dcn, biglycan (Bgn), and versican (Vcan), which are core proteins of CS/DS-PGs (Fig 3D).

Download:

Fig 3. Pathological findings in Chst14^-/- mice and changes in collagen fibrils due to DS deficiency.

(A) H&E- and Sirius red-stained colon sections from Chst14^+/+ and Chst14^-/- mice. Scale bar: 200 µm. Longitudinal and transverse sections of collagen fibrils in the submucosa of the colon observed by TEM. Scale bar: 500 nm. Collagen fibrils in the submucosa of the colon observed by SEM. Scale bar: 1 µ m. (B) Colon layer thickness. Chst14^+/+ and Chst14^-/- mice, n = 8 per group. (C) Comparison of collagen diameter (mean ± SEM). Chst14^+/+ and Chst14^-/- mice, n = 3 per group. ^*P < 0.05, t-test. Histogram of collagen fibril diameter. White bars: collagen fibrils in Chst14^+/+ mice, n = 1306; and gray bars: collagen fibrils in Chst14^-/- mice, n = 806. (D) Relative mRNA levels of Col1a1, Col3a1, Dcn, Bgn, and Vcan in the colon (mean ± SEM) analyzed by t-test. Chst14^+/+ and Chst14^-/- mice, n = 6 per group. Chst14^+/+ mice, + / + ; Chst14^-/- mice, -/-.

https://doi.org/10.1371/journal.pone.0320943.g003

Chst14^-/- mice do not have more severe DSS-induced colitis compared with Chst14^+/+ mice

The DSS-induced colitis model was used to investigate the inflammatory response provoked by intestinal mucosal damage. No difference was observed in the amount of DSS-containing water consumed by Chst14^-/- and Chst14^+/+ mice (Fig 4A). The DSS administration model is known to induce weight loss, loose stools, diarrhea, and bloody stools [36]. There were no significant differences between Chst14^-/- and Chst14^+/+ mice in DAI scores and body weight changes (Fig 4B, C). Weight loss peaked on day 10 (2 days after termination of DSS administration) and then recovered in both groups (Fig 4C). On day 8 of DSS administration, significant colon shortening was observed in Chst14^+/+ mice compared with the control group. This shortening was not observed in Chst14^-/- mice (Fig 4D, E). H&E staining on day 8 of DSS administration showed destruction of glandular structures, ulceration of the mucosal layer, marked edema of the submucosal layer, and infiltration of inflammatory cells throughout all layers in both Chst14^-/- and Chst14^+/+ mice (Fig 4G). H&E staining on day 15 after the start of DSS administration (7 days after termination of DSS administration) showed improvements in edema in both groups (Fig 4G). However, Chst14^+/+ mice showed more persistent inflammatory cell infiltration and destruction of the mucosal layer than Chst14^-/- mice (Fig 4G). Based on H&E scores, there was no significant difference in colon tissue injury between Chst14^-/- and Chst14^+/+ mice at day 8. In contrast, Chst14^-/- mice exhibited a significantly lower H&E score compared with Chst14^+/+ mice on day 15 (Fig 4F). Alcian blue staining, which detects mucins and GAGs present in the intestinal mucosal glands, identified destroyed glandular structures and decreased mucin on day 8 in both Chst14^-/- and Chst14^+/+ mice (Fig 4G). By day 15, Chst14^-/- mice showed greater amounts of mucin and/or GAGs compared with Chst14^+/+ mice (Fig 4G). Sirius red staining was diffuse in the disrupted mucosal layer of both Chst14^-/- and Chst14^+/+ mice on day 8 of DSS administration (Fig 4G). On day 15, Chst14^+/+ mice exhibited stronger Sirius red staining in the submucosa, indicating increased collagen hyperplasia compared with Chst14^-/- mice (Fig 4G). TEM showed disorganized collagen fibril assembly in the submucosa of both Chst14^-/- and Chst14^+/+ mice on day 8 of DSS administration (Fig 4G). On day 15, collagen fibrils appeared to reassemble in Chst14^+/+ mice (Fig 4G).

Download:

Fig 4. Experimental course and inflammatory response in the DSS colitis model.

(A) Intake of DSS solution (mean ± SEM). Volume of DSS solution was adjusted using a specific gravity of 1.016. (B) Change in DAI scores (mean ± SEM). (C) Percentage changes in body weight (mean ± SEM). (B, C) Black circle: Chst14^+/+ mice; and white triangle: Chst14^-/- mice. D1–D8: Chst14^+/+ mice, n = 16; and Chst14^-/- mice, n = 14; D9–D15: Chst14^+/+ mice, n = 8; and Chst14^-/- mice, n = 7. (D) Typical colon appearance in Chst14^+/+ and Chst14^-/- mice under control conditions, 2.0% DSS (day 8), and 2.0% DSS (day 15). (E) Comparison of colon length (mean ± SEM). (F) Comparison of H&E scores to examine colon injury (mean ± SEM). (A, E, F) Chst14^+/+ mice, n = 8; and Chst14^-/- mice, n = 7. Data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test. (E) ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001 compared with Chst14^+/+ control group. ^†P < 0.05, ^††P < 0.01 compared with 2.0% DSS (day 8) Chst14^+/+ group. ^‡‡P < 0.01 compared with 2.0% DSS (day 8) Chst14^-/- group. (F) ^**P < 0.01, ^****P < 0.0001 compared with Chst14^+/+control group. ^##P < 0.01, ^####P < 0.0001 compared with Chst14^-/- control group. ^†P < 0.05 compared with 2.0% DSS (day 8) Chst14^+/+ group. ^§P < 0.05 compared with 2.0% DSS (day 15) Chst14^+/+ group. (G) Colon samples stained with H&E, Alcian blue, and Sirius red under control, 2.0% DSS (day 8), and 2.0% DSS (day 15) conditions. Scale bar: 200 µm. TEM observation of control and 2.0% DSS (day 8 and 15) groups. Scale bar: 500 nm. Cont., control; D8, day 8; D15, day 15; DAI, disease activity index; Chst14^+/+ mice, + / + ; Chst14^-/- mice, -/-.

https://doi.org/10.1371/journal.pone.0320943.g004

Inflammatory response in DSS-induced colitis in DS deficiency mice

Immunostaining for Ly-6G was used to examine neutrophil infiltration. On day 8, the number of Ly-6G-positive cells was similar between Chst14^-/- and Chst14^+/+ mice (Fig 5A, B). On day 15, Chst14^-/- mice tended to have fewer neutrophils compared with Chst14^+/+ mice (Fig 5A, B). Gene expression of transforming growth factor beta (Tgfb) and tumor necrosis factor-alpha (Tnfa) were significantly increased on day 8 in both Chst14^-/- and Chst14^+/+ mice (Fig 5C). After discontinuing DSS administration (day 15), gene expression of Tgfb was decreased in Chst14^-/- mice only, and continued to rise in Chst14^+/+ mice. Gene expression of Tnfa decreased in both Chst14^-/- and Chst14^+/+ mice on day 15 (Fig 5C). Notably, gene expression of interleukin 1 beta (Il1b) increased significantly in Chst14^+/+ mice only on day 8 (Fig 5C).

Download:

Fig 5. Fluorescent immunostaining of Ly-6G and gene expression of inflammation-related factors.

(A) Representative results of fluorescent immunostaining of Ly-6G, DAPI, and merged images. (B) Comparison of the number of Ly-6G-positive cells. Values for each individual point reflect the average of three measurements from all fields of a section. Control group – Chst14^+/+ and Chst14^-/- mice, n = 5 per group; 2.0% DSS (day 8) group – Chst14^+/+ and Chst14^-/- mice, n = 5 per group; 2.0% DSS (day 15) group – Chst14^+/+ mice, n = 6; and Chst14^-/- mice, n = 5. Mean ± SEM. (C) Relative mRNA levels of Tnfa, Il1b, and Tgfb in the colon of DSS-induced colitis model mice. Chst14^+/+ and Chst14^-/- mice, n = 6 per group. Mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with control Chst14^+/+ group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with control Chst14^-/- group. ^†P < 0.05, ^††P < 0.01 compared with 2.0% DSS (day 8) Chst14^+/+ group. ^‡P < 0.05 compared with 2.0% DSS (day 8) Chst14^-/- group. Cont., control; D8, day 8; D15, day 15; Chst14^+/+ mice, + / + ; Chst14^-/- mice, -/-.

https://doi.org/10.1371/journal.pone.0320943.g005

Discussion

In this study, we sought to characterize the colonic phenotype of Chst14^-/- mice to investigate the effects of DS deficiency. The colons of Chst14^-/- mice exhibited a loss of DS disaccharides, shortening due to increased contractility, smaller collagen fibril diameters, and abnormal submucosal collagen fibril networks. There were no significant differences in physiological indicators and colon strength or extensibility. DSS colitis in Chst14^-/- mice showed no aggravation compared with Chst14^+/+ mice. These findings may enhance our understanding of DS function in the colon.

Neither DS nor Chst14 gene expression were detected in the colons of Chst14^-/- mice. On the other hand, CS synthesis was elevated in the colon compared with Chst14^+/+ mice. The compensatory increase in CS was thought to be due to accumulation of dermatan caused by loss of D4ST1 and promotion of chondroitin synthesis with reverse isomerization by DSE [37,38]. Aside from Chst14, the gene expression of other enzymes associated with CS/DS synthesis was similar in the colons of Chst14^-/- and Chst14^+/+ mice. These results indicate that the compensatory increase in CS in the colon of Chst14^-/- mice occurs within the normal range of gene expression of relevant CS/DS synthetic enzymes.

H&E staining of colon tissue from Chst14^-/- mice showed no obvious pathological differences, as previously reported in humans [17]. However, electron microscopy detected poorly aggregated collagen fibrils with reduced density and smaller diameters in the submucosa of Chst14^-/- mouse colon. Collagen fibrils in the colon are composed of a hybrid of type 1 and 3 collagens [39], with collagen diameter affected by the proportion of type 3 collagen [40]. However, we found no differences in gene expression of molecules related to collagen fibrils (Col1a1 and Col3a1) or core proteins of proteoglycans (Dcn, Bgn, and Vcan) between Chst14^-/- and Chst14^+/+ mice. These results suggest that morphological changes in collagen fibrils in the colon of Chst14^-/- mice are not caused by quantitative changes in either collagen molecules or the core proteins of CS/DS-PGs. Interactions between CS/DS-PGs and collagen are reportedly involved in collagen fibril assembly [24]. In the skin of healthy individuals and Chst14^+/+ mice, CS/DS GAG chains are curved and in close contact along the contours of attached collagen fibrils, whereas in patients with mcEDS-CHST14 and Chst14^-/- mice, the CS-only GAG chains are linear [22]. Biochemical alterations in GAGs resulting from the lack of DS lead to defective assembly of collagen fibrils in the skin [22]. DCN is known as a core protein of CS/DS-PG that binds to type 1 collagen [41]. In this study, DCN was identified as a core protein of CS/DS in the colon. Furthermore, DS on DCN in the colon was significantly reduced and replaced with CS in the absence of Chst14. Therefore, it was suggested that the mechanism of defective collagen fibril assembly in the colon is similar to that in the skin.

Furthermore, deformation of collagen fibril assembly is regarded as a cause of skin fragility in patients with mcEDS and Chst14^-/- mice [22,24]. In the colon, collagen fibrils are localized in the submucosa and are thought to provide structural strength [42]. In the present study, we found no differences in tissue strength or extensibility, and no signs of tissue fragility in the colon. Unlike in the skin, colon tissue contains muscles that run in multiple directions [43]. Therefore, a limitation of this study is that it may have only evaluated the strength and extensibility of the colon in one direction. The muscular layer of the colon may compensate for tissue fragility caused by structural changes in submucosal collagen fibrils.

Colons were shorter in Chst14^-/- mice compared with Chst14^+/+ mice. We investigated reasons for this shortening of the colon. Administration of the calcium channel blocker, nifedipine (which is also a smooth muscle relaxant), eliminated the difference in colon length between Chst14^-/- and Chst14^+/+ mice. This suggests that colon length in Chst14^-/- mice is influenced by intestinal contraction. Intestinal contraction is regulated by smooth muscle, nerves, ion channels, and cell adhesion. However, no differences were detected in the gene expression of the factors related to colonic smooth muscle contraction (S1 Fig A). There is a possibility that the structural changes in collagen fibril assembly influence intestinal contraction, though further research is necessary. We also hypothesized that DS might affect the physiological function of the colon in Chst14^-/- mice. However, Chst14^-/- mice showed no differences in food intake, defecation volume, gastrointestinal transit time, and rectoanal reflex. No differences in peristalsis were detected by abdominal ultrasound between Chst14^+/+ and Chst14^-/- mice (S1, S2 Movies, S4 Fig A, B). These data indicate that there are no significant differences in the physiological function of the colon in these mice. Unlike patients with mcEDS-CHST14, constipation was not detected in Chst14^-/- mice. This may be influenced by the fact that mice do not withhold defecation and lack a sigmoid colon to store stool [43]. Additionally, based on macroscopic and pathological analyses of the colon, we found no diverticula in Chst14^-/- mice. Diverticula are believed to develop when the colon tissue weakens and intraluminal pressure increases [44,45], causing the mucosa and submucosa to protrude through the muscularis propria, forming sac-like structures [46]. Diverticula are frequently observed along the tenia coli, which are vulnerable sites for diverticula formation in humans [46,47]. However, mice do not have this structure [48], which along with the absence of these corresponding risk factors, may partially explain the reduced likelihood of diverticula formation.

Age-dependent increases in body weight and colon length were observed in middle-aged Chst14^+/+ and Chst14^-/- mice compared with young mice (S2 Fig A–C). The gene expression of Col1a1 was decreased in middle-aged Chst14^+/+ and Chst14^-/- mice (S2 Fig J). Similar to young mice, middle-aged Chst14^-/- mice also exhibited colon shortening compared with age-matched Chst14^+/+ mice (S2 Fig A, C). However, no age-related phenotypic changes were observed in the colons of middle-aged Chst14^-/- mice (S2 Fig D–I).

Female Chst14^-/- mice also showed reduced body weight and colon shortening, similar to male mice (S3 Fig A–C). Furthermore, nifedipine suppressed intestinal contractions and eliminated the difference in colon length between Chst14^+/+ and Chst14^-/- mice (S3 Fig D). On the other hand, gene expression of Dcn, collagen-related factor, was significantly decreased in female Chst14^-/- mice compared with that in male Chst14^+/+ and Chst14^-/- mice (S3 Fig F). Detailed examination of the physiological functions of the colon in female Chst14^-/- mice is a subject for future research.

Some patients with mcEDS-CHST14 experience diverticulitis and secondary diverticular perforation [16]. Constipation and increased intestinal pressure are contributing factors to developing diverticulitis, while the mechanisms of inflammation and healing associated with infection and ischemia play critical roles in its progression [49,50]. DS has been reported to interact with growth factors and is associated with wound healing in vitro [51]. We next hypothesized that inflammation would be more severe in Chst14^-/- mice. As Chst14^-/- mice did not develop diverticula, the primary cause of diverticulitis, we used the DSS-induced colitis model, a widely recognized model for inflammatory bowel disease, to examine whether DS deficiency influences the severity of inflammation or the healing process [36]. However, under the same dose of DSS intake, there was no difference in the progression of colitis between Chst14^-/- and Chst14^+/+ mice. Colon shortening is one of the findings associated with inflammation and is observed in patients with ulcerative colitis and in the DSS colitis model [28]. Significant colon shortening was observed in Chst14^+/+ mice but not Chst14^-/- mice. Tissue damage was examined by several histological analyses such as H&E, Alcian blue, and Sirius red staining, and TEM. No analyses revealed evidence of more severe inflammation in Chst14^-/- mice compared with Chst14^+/+ mice. Indeed, H&E score suggested that Chst14^-/- mice showed faster histological repair of DSS-induced colitis damage compared with Chst14^+/+ mice. Additionally, Ly-6G immunostaining was performed to examine neutrophil infiltration, but no significant differences were observed. Furthermore, gene expression of inflammatory factors such as Tgfb, Tnfa, and Il1b, showed no significant differences between Chst14^-/- and Chst14^+/+ mice [52–54]. These results suggest that DS deficiency does not aggravate inflammation and healing in the mouse colon.

Conclusions

Functions of DS in the colon have not been previously elucidated. In this study, we found that DS deficiency affects collagen assembly and intestinal contraction. However, unlike humans, no significant alterations in physiological function of the colon were observed in mouse. While further detailed investigations are required to fully understand the effects of DS on gastrointestinal physiological functions, this study sheds light on the role of DS in the colon and the potential mechanisms underlying gastrointestinal symptoms in patients with mcEDS.

Supporting information

S1 Fig. Gene expression levels related to colonic smooth muscle contraction.

(A) Relative mRNA levels (mean ± SEM) of Acta2, Myh11, Mylk, Rock1, Ryr2, Kcnq1, Cacna1c, Cnn1, Calm1, Ano1, Adrb1, Adrb2, Chrm2, Chrm3, Chat, Htr1a, Htr2a, Htr2b, Htr3a, Htr3b, Htr4, Htr7, Cx26, and Gja1 in colon samples from 10-week-old Chst14^+/+ and Chst14^-/- mice (n = 6 per group). Gene abbreviations: Acta2, actin alpha 2; Myh11, myosin heavy chain 11; Mylk, myosin light chain kinase; Rock1, Rho-associated cooled containing protein kinase 1; Ryr2, ryanodine receptor 2; Kcnq1, potassium voltage-gated channel subfamily Q member 1; Cacna1c, calcium channel voltage-dependent L type alpha 1C subunit; Cnn1, calponin 1; Calm1, calmodulin 1; Ano1, calcium activated chloride channel; Adrb1, adrenergic receptor, beta 1; Adrb2, adrenergic receptor, beta 2; Chrm2, cholinergic receptor, muscarinic 2; Chrm3, cholinergic receptor, muscarinic 3; Chat, choline acetyltransferase; Htr1a, 5-hydroxytryptamine receptor 1A; Htr2a, 5-hydroxytryptamine receptor 2A; Htr2b, 5-hydroxytryptamine receptor 2B; Htr3a, 5-hydroxytryptamine receptor 3A; Htr3b, 5-hydroxytryptamine receptor 3B; Htr4, 5-hydroxytryptamine receptor 4; Htr7, 5-hydroxytryptamine receptor 7; Cx26, connexin-26; Gja1, gap junction protein alpha 1.

https://doi.org/10.1371/journal.pone.0320943.s001

(PDF)

S2 Fig. Colonic morphology, function, and collagen-related gene expression in middle-aged mice.

(A) Typical appearance of the colon in middle-aged mice. (B) Body weight (mean ± SEM). (C) Comparison of colon length between young and middle-aged groups (mean ± SEM). (B), (C) Young Chst14^+/+ mice, n = 7; and Chst14^-/- mice, n = 7; and middle-aged Chst14^+/+ mice, n = 8; and Chst14^-/- mice, n = 9. Data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with young Chst14^+/+ group. ^##P < 0.01, ^####P < 0.0001 compared with young the Chst14^-/- group. ^†P < 0.05, ^††††P < 0.0001 compared with middle-aged Chst14^+/+ group. (D) H&E-stained colon sections from middle-aged Chst14^+/+ and Chst14^-/- mice. Scale bar: 200 µm. (E) Amount of defecation per day. (F) Daily food intake. (E, F) Young Chst14^+/+ mice, n = 8; and young Chst14^-/- mice, n = 8; and middle-aged Chst14^+/+ mice, n = 8; and middle-aged Chst14^-/- mice, n = 9. (G) Load required for colon rupture. (H) Rate of colon extension when loaded with a 10 g weight. (G, H) Young Chst14 ^+/+ mice, n = 12; and young Chst14^-/- mice, n = 6; and middle-aged Chst14^+/+ mice, n = 8; and middle-aged Chst14^-/- mice, n = 9. (I) Gastrointestinal transit time. Young Chst14^+/+ mice, n = 9; and young Chst14^-/- mice, n = 8; and middle-aged Chst14^+/+ mice, n = 8; and middle-aged Chst14^-/- mice, n = 9. (J) Relative mRNA levels of Col1a1, Col3a1, Dcn, Bgn, and Vcan in the colon (mean ± SEM) analyzed by one-way ANOVA followed by Tukey–Kramer post hoc test. Young and middle-aged Chst14^+/+ and Chst14^-/- mice (n = 6 per group). Young mice, 10-week-old male mice; middle-aged mice, 36- to 40-week-old male mice. Chst14^+/+ mice, + / + ; Chst14^-/- mice, -/-.

https://doi.org/10.1371/journal.pone.0320943.s002

(PDF)

S3 Fig. Colonic morphology and collagen-related gene expression in female mice.

(A) Typical appearance of the colon in female mice. (B) Comparison of body weight between female Chst14^+/+ and Chst14^-/- mice. (mean ± SEM). ^***P < 0.001 compared with Chst14^+/+. (C) Comparison of colon length between female Chst14^+/+ and Chst14^-/- mice. (mean ± SEM). ^*P < 0.05 compared with Chst14^+/+. (B, C) Chst14^+/+ mice, n = 5 and Chst14^-/- mice, n = 3. Data were analyzed using t-test. (D) Comparison of colon length between pre- and post-nifedipine groups (mean ± SEM). Pre- and post-nifedipine Chst14^+/+ mice (n = 5) and Chst14^-/- mice (n = 3). Data were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc test. ^*P < 0.05 compared with pre-nifedipine Chst14^+/+ group. ^##P < 0.001 compared with pre-nifedipine Chst14^-/- group. (E) H&E-stained colon sections from Chst14^+/+ and Chst14^-/- mice. Scale bar: 200 µm. (F) Relative mRNA levels of Col1a1, Col3a1, Dcn, Bgn, and Vcan in the colon (mean ± SEM) analyzed by one-way ANOVA followed by Tukey–Kramer post hoc test. Male and female Chst14^+/+ and Chst14^-/- mice (n = 6 per group). ^*P < 0.05 compared with male Chst14^+/+ group. ^#P < 0.05 compared with male Chst14^-/- group. Chst14^+/+ mice, + / + ; Chst14^-/- mice, -/-; Male, 10-week-old male mice; female, 12-week-old female mice.

https://doi.org/10.1371/journal.pone.0320943.s003

(PDF)

S4 Fig. Still images extracted from ultrasound videos showing the colons of Chst14^+/+ and Chst14^-/- mice.

https://doi.org/10.1371/journal.pone.0320943.s004

(PDF)

S1 Movie. A representative ultrasound video of the colon from a Chst14^+/+ mouse.

Abdominal ultrasound was performed for more than 10 minutes on each of the five male Chst14^+/+ mice (11 weeks old), and a representative 8-second video was obtained.

https://doi.org/10.1371/journal.pone.0320943.s005

(MP4)

S2 Movie. A representative ultrasound video of the colon from a Chst14^-/- mouse.

Abdominal ultrasound was performed for more than 10 minutes on each of the three male Chst14^-/- mice (11–12 weeks old), and a representative 8-second video was obtained.

https://doi.org/10.1371/journal.pone.0320943.s006

(MP4)

S1 Table. RT-qPCR primer sequences.

https://doi.org/10.1371/journal.pone.0320943.s007

(PDF)

S2 Table. Relative mRNA levels.

https://doi.org/10.1371/journal.pone.0320943.s008

(PDF)

S3 Table. Disaccharide composition of CS/DS, CS, and DS.

https://doi.org/10.1371/journal.pone.0320943.s009

(PDF)

S4 Table. Physiological measurements.

https://doi.org/10.1371/journal.pone.0320943.s010

(PDF)

S5 Table. Measured thicknesses of each layer.

https://doi.org/10.1371/journal.pone.0320943.s011

(PDF)

S6 Table. Measured diameter of collagen fibrils.

https://doi.org/10.1371/journal.pone.0320943.s012

(PDF)

S7 Table. Data of DSS colitis model.

https://doi.org/10.1371/journal.pone.0320943.s013

(PDF)

S8 Table. Number of Ly-6G-positive cells.

https://doi.org/10.1371/journal.pone.0320943.s014

(PDF)

S1 File. Raw images.

Uncropped Western blot images for DCN and GAPDH.

https://doi.org/10.1371/journal.pone.0320943.s015

(PDF)

Acknowledgments

We are grateful to the Division of Instrumental Research, Research Center for Advanced Science and Technology, Shinshu University, for its facilities and scientific and technical support. We thank all the staff of the Division of Animal Research, Research Center for Advanced Science and Technology, Shinshu University for their cooperation in the experiments. The authors thank Alison McTavish, MSc from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

References

1. Mizumoto S, Yamada S. The Specific Role of Dermatan Sulfate as an Instructive Glycosaminoglycan in Tissue Development. Int J Mol Sci. 2022;23(13).
- View Article
- Google Scholar
2. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem. 1998;67:609–52. pmid:9759499
- View Article
- PubMed/NCBI
- Google Scholar
3. Trowbridge JM, Gallo RL. Dermatan sulfate: new functions from an old glycosaminoglycan. Glycobiology. 2002;12(9):117R-25R. pmid:12213784
- View Article
- PubMed/NCBI
- Google Scholar
4. Mizumoto S, Yamada S, Sugahara K. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins. Curr Opin Struct Biol. 2015;34:35–42. pmid:26164146
- View Article
- PubMed/NCBI
- Google Scholar
5. Malmström A. Biosynthesis of dermatan sulphate. Loss of C-5 hydrogen during conversion of D-glucuronate to L-iduronate. Biochem J. 1981;198(3):669–75. pmid:7326032
- View Article
- PubMed/NCBI
- Google Scholar
6. Pacheco B, Maccarana M, Malmström A. Dermatan 4-O-sulfotransferase 1 is pivotal in the formation of iduronic acid blocks in dermatan sulfate. Glycobiology. 2009;19(11):1197–203. pmid:19661164
- View Article
- PubMed/NCBI
- Google Scholar
7. Mizumoto S, Yamada S. Histories of Dermatan Sulfate Epimerase and Dermatan 4-O-Sulfotransferase from Discovery of Their Enzymes and Genes to Musculocontractural Ehlers-Danlos Syndrome. Genes (Basel). 2023;14:2.
- View Article
- Google Scholar
8. Malmström A, Fransson LA. Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues. J Biol Chem. 1975;250(9):3419–25.
- View Article
- Google Scholar
9. Dündar M, Muller T, Zhang Q, Pan J, Steinmann B, Vodopiutz J, et al. Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome. Am J Hum Genet. 2009;85(6):873–82.
- View Article
- Google Scholar
10. Miyake N, Kosho T, Mizumoto S, Furuichi T, Hatamochi A, Nagashima Y, et al. Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome. Hum Mutat. 2010;31(8):966–74. pmid:20533528
- View Article
- PubMed/NCBI
- Google Scholar
11. Müller T, Mizumoto S, Suresh I, Komatsu Y, Vodopiutz J, Dundar M, et al. Loss of dermatan sulfate epimerase (DSE) function results in musculocontractural Ehlers-Danlos syndrome. Hum Mol Genet. 2013;22(18):3761–72. pmid:23704329
- View Article
- PubMed/NCBI
- Google Scholar
12. Malfait F, Francomano C, Byers P, Belmont J, Berglund B, Black J, et al. The 2017 international classification of the Ehlers-Danlos syndromes. Am J Med Genet C Semin Med Genet. 2017;175(1):8–26. pmid:28306229
- View Article
- PubMed/NCBI
- Google Scholar
13. Mizumoto S, Kosho T, Hatamochi A, Honda T, Yamaguchi T, Okamoto N, et al. Defect in dermatan sulfate in urine of patients with Ehlers-Danlos syndrome caused by a CHST14/D4ST1 deficiency. Clin Biochem. 2017;50(12):670–7. pmid:28238810
- View Article
- PubMed/NCBI
- Google Scholar
14. Minatogawa M, Hirose T, Mizumoto S, Yamaguchi T, Nagae C, Taki M, et al. Clinical and pathophysiological delineation of musculocontractural Ehlers-Danlos syndrome caused by dermatan sulfate epimerase deficiency (mcEDS-DSE): A detailed and comprehensive glycobiological and pathological investigation in a novel patient. Hum Mutat. 2022;43(12):1829–36. pmid:35842784
- View Article
- PubMed/NCBI
- Google Scholar
15. Brady AF, Demirdas S, Fournel-Gigleux S, Ghali N, Giunta C, Kapferer-Seebacher I, et al. The Ehlers-Danlos syndromes, rare types. Am J Med Genet C Semin Med Genet. 2017;175(1):70–115.
- View Article
- Google Scholar
16. Minatogawa M, Unzaki A, Morisaki H, Syx D, Sonoda T, Janecke AR, et al. Clinical and molecular features of 66 patients with musculocontractural Ehlers-Danlos syndrome caused by pathogenic variants in CHST14 (mcEDS-CHST14). J Med Genet. 2022;59(9):865–77. pmid:34815299
- View Article
- PubMed/NCBI
- Google Scholar
17. Kobayashi T, Fujishima F, Tokodai K, Sato C, Kamei T, Miyake N, et al. Detailed Courses and Pathological Findings of Colonic Perforation without Diverticula in Sisters with Musculocontractural Ehlers-Danlos Syndrome Caused by Pathogenic Variant in CHST14 (mcEDS-CHST14). Genes. 2023;14(5).
- View Article
- Google Scholar
18. Qian H, Zhou T, Zheng N, Lu Q, Han Y. Case report: Multiple gastrointestinal perforations in a rare musculocontractural Ehlers-Danlos syndrome with multiple organ dysfunction. Front Genet. 2022;13:846529. pmid:36046248
- View Article
- PubMed/NCBI
- Google Scholar
19. Bian S, Akyüz N, Bernreuther C, Loers G, Laczynska E, Jakovcevski I, et al. Dermatan sulfotransferase Chst14/D4st1, but not chondroitin sulfotransferase Chst11/C4st1, regulates proliferation and neurogenesis of neural progenitor cells. J Cell Sci. 2011;124(Pt 23):4051–63. pmid:22159417
- View Article
- PubMed/NCBI
- Google Scholar
20. Nitahara-Kasahara Y, Mizumoto S, Inoue YU, Saka S, Posadas-Herrera G, Nakamura-Takahashi A, et al. A new mouse model of Ehlers-Danlos syndrome generated using CRISPR/Cas9-mediated genomic editing. Dis Model Mech. 2021;14(12):dmm048963. pmid:34850861
- View Article
- PubMed/NCBI
- Google Scholar
21. Akyüz N, Rost S, Mehanna A, Bian S, Loers G, Oezen I, et al. Dermatan 4-O-sulfotransferase1 ablation accelerates peripheral nerve regeneration. Exp Neurol. 2013;247:517–30.
- View Article
- Google Scholar
22. Hirose T, Mizumoto S, Hashimoto A, Takahashi Y, Yoshizawa T, Nitahara-Kasahara Y, et al. Systematic investigation of the skin in Chst14-/- mice: A model for skin fragility in musculocontractural Ehlers-Danlos syndrome caused by CHST14 variants (mcEDS-CHST14). Glycobiology. 2021;31(2):137–50. pmid:32601684
- View Article
- PubMed/NCBI
- Google Scholar
23. Yoshizawa T, Mizumoto S, Takahashi Y, Shimada S, Sugahara K, Nakayama J, et al. Vascular abnormalities in the placenta of Chst14-/- fetuses: implications in the pathophysiology of perinatal lethality of the murine model and vascular lesions in human CHST14/D4ST1 deficiency. Glycobiology. 2018;28(2):80–9. pmid:29206923
- View Article
- PubMed/NCBI
- Google Scholar
24. Hirose T, Takahashi N, Tangkawattana P, Minaguchi J, Mizumoto S, Yamada S, et al. Structural alteration of glycosaminoglycan side chains and spatial disorganization of collagen networks in the skin of patients with mcEDS-CHST14. Biochim Biophys Acta Gen Subj. 2019;1863(3):623–31. pmid:30553867
- View Article
- PubMed/NCBI
- Google Scholar
25. Tang T, Li L, Tang J, Li Y, Lin WY, Martin F, et al. A mouse knockout library for secreted and transmembrane proteins. Nat Biotechnol. 2010;28(7):749–55.
- View Article
- Google Scholar
26. Shimada S, Yoshizawa T, Takahashi Y, Nitahara-Kasahara Y, Okada T, Nomura Y, et al. Backcrossing to an appropriate genetic background improves the birth rate of carbohydrate sulfotransferase 14 gene-deleted mice. Exp Anim. 2020;69(4):407–13. pmid:32522905
- View Article
- PubMed/NCBI
- Google Scholar
27. Lackey AI, Chen T, Zhou YX, Bottasso Arias NM, Doran JM, Zacharisen SM, et al. Mechanisms underlying reduced weight gain in intestinal fatty acid-binding protein (IFABP) null mice. Am J Physiol Gastrointest Liver Physiol. 2020;318(3):G518–30. pmid:31905021
- View Article
- PubMed/NCBI
- Google Scholar
28. Chassaing B, Aitken JD, Malleshappa M, Vijay-Kumar M. Dextran sulfate sodium (DSS)-induced colitis in mice. Curr Protoc Immunol. 2014;104:15.25.1-15.25.14. pmid:24510619
- View Article
- PubMed/NCBI
- Google Scholar
29. Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993;69(2):238–49.
- View Article
- Google Scholar
30. Ghia J-E, Blennerhassett P, Collins SM. Impaired parasympathetic function increases susceptibility to inflammatory bowel disease in a mouse model of depression. J Clin Invest. 2008;118(6):2209–18. pmid:18451995
- View Article
- PubMed/NCBI
- Google Scholar
31. Hiura K, Maruyama T, Watanabe M, Nakano K, Okamura T, Sasaki H, et al. Mitotic spindle positioning protein (MISP) deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice. J Vet Med Sci. 2023;85(2):167–74. pmid:36596561
- View Article
- PubMed/NCBI
- Google Scholar
32. Dieleman LA, Palmen MJ, Akol H, Bloemena E, Pena AS, Meuwissen SG, Van Rees EP. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 1998;114(3):385–91.
- View Article
- Google Scholar
33. Jialing L, Yangyang G, Jing Z, Xiaoyi T, Ping W, Liwei S, et al. Changes in serum inflammatory cytokine levels and intestinal flora in a self-healing dextran sodium sulfate-induced ulcerative colitis murine model. Life Sci. 2020;263:118587. pmid:33065145
- View Article
- PubMed/NCBI
- Google Scholar
34. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. pmid:22743772
- View Article
- PubMed/NCBI
- Google Scholar
35. Mizumoto S, Sugahara K. Glycosaminoglycan chain analysis and characterization (glycosylation/epimerization). Methods Mol Biol. 2012;836:99–115. pmid:22252630
- View Article
- PubMed/NCBI
- Google Scholar
36. Eichele DD, Kharbanda KK. Dextran sodium sulfate colitis murine model: An indispensable tool for advancing our understanding of inflammatory bowel diseases pathogenesis. World J Gastroenterol. 2017;23(33):6016–29. pmid:28970718
- View Article
- PubMed/NCBI
- Google Scholar
37. Hannesson HH, Hagner-McWhirter A, Tiedemann K, Lindahl U, Malmstrom A. Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism. Biochem J. 1996;313(Pt 2):589–96.
- View Article
- Google Scholar
38. Maccarana M, Olander B, Malmström J, Tiedemann K, Aebersold R, Lindahl U, et al. Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5-epimerase is identical to SART2. J Biol Chem. 2006;281(17):11560–8. pmid:16505484
- View Article
- PubMed/NCBI
- Google Scholar
39. Fleischmajer R, Perlish JS, Burgeson RE, Shaikh-Bahai F, Timpl R. Type I and type III collagen interactions during fibrillogenesis. Ann N Y Acad Sci. 1990;580:161–75. pmid:2186689
- View Article
- PubMed/NCBI
- Google Scholar
40. Stuart K, Panitch A. Characterization of gels composed of blends of collagen I, collagen III, and chondroitin sulfate. Biomacromolecules. 2009;10(1):25–31. pmid:19053290
- View Article
- PubMed/NCBI
- Google Scholar
41. Keene DR, San Antonio JD, Mayne R, McQuillan DJ, Sarris G, Santoro SA, et al. Decorin binds near the C terminus of type I collagen. J Biol Chem. 2000;275(29):21801–4. pmid:10823816
- View Article
- PubMed/NCBI
- Google Scholar
42. Maier F, Siri S, Santos S, Chen L, Feng B, Pierce DM. The heterogeneous morphology of networked collagen in distal colon and rectum of mice quantified via nonlinear microscopy. J Mech Behav Biomed Mater. 2021;113:104116. pmid:33049619
- View Article
- PubMed/NCBI
- Google Scholar
43. Durcan C, Hossain M, Chagnon G, Perić D, Girard E. Mechanical experimentation of the gastrointestinal tract: a systematic review. Biomech Model Mechanobiol. 2024;23(1):23–59. pmid:37935880
- View Article
- PubMed/NCBI
- Google Scholar
44. Feuerstein JD, Falchuk KR. Diverticulosis and Diverticulitis. Mayo Clin Proc. 2016;91(8):1094–104.
- View Article
- Google Scholar
45. Tursi A. Current and Evolving Concepts on the Pathogenesis of Diverticular Disease. J Gastrointestin Liver Dis. 2019;28:225–35. pmid:31204408
- View Article
- PubMed/NCBI
- Google Scholar
46. Wedel T, Barrenschee M, Lange C, Cossais F, Böttner M. Morphologic Basis for Developing Diverticular Disease, Diverticulitis, and Diverticular Bleeding. Viszeralmedizin. 2015;31(2):76–82. pmid:26989376
- View Article
- PubMed/NCBI
- Google Scholar
47. Barbaro MR, Cremon C, Fuschi D, Marasco G, Palombo M, Stanghellini V, Barbara G. Pathophysiology of Diverticular Disease: From Diverticula Formation to Symptom Generation. Int J Mol Sci. 2022;23(12).
- View Article
- Google Scholar
48. Patel B, Guo X, Noblet J, Chambers S, Kassab GS. Animal Models of Diverticulosis: Review and Recommendations. Dig Dis Sci. 2018;63(6):1409–18.
- View Article
- Google Scholar
49. Zullo A. Medical hypothesis: speculating on the pathogenesis of acute diverticulitis. Ann Gastroenterol. 2018;31(6):747–9.
- View Article
- Google Scholar
50. Piscopo N, Ellul P. Diverticular Disease: A Review on Pathophysiology and Recent Evidence. Ulster Med J. 2020;89(2):83–8. pmid:33093692
- View Article
- PubMed/NCBI
- Google Scholar
51. Taylor KR, Rudisill JA, Gallo RL. Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity. J Biol Chem. 2005;280(7):5300–6. pmid:15563459
- View Article
- PubMed/NCBI
- Google Scholar
52. Stevens C, Walz G, Singaram C, Lipman ML, Zanker B, Muggia A, et al. Tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 expression in inflammatory bowel disease. Dig Dis Sci. 1992;37(6):818–26.
- View Article
- Google Scholar
53. Al-Sadi R, Boivin M, Ma T. Mechanism of cytokine modulation of epithelial tight junction barrier. Front Biosci (Landmark Ed). 2009;14(7):2765–78. pmid:19273235
- View Article
- PubMed/NCBI
- Google Scholar
54. Biancheri P, Giuffrida P, Docena GH, MacDonald TT, Corazza GR, Di Sabatino A. The role of transforming growth factor (TGF)-β in modulating the immune response and fibrogenesis in the gut. Cytokine Growth Factor Rev. 2014;25(1):45–55. pmid:24332927
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Mizumoto S, Yamada S. The Specific Role of Dermatan Sulfate as an Instructive Glycosaminoglycan in Tissue Development. Int J Mol Sci. 2022;23(13).
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem. 1998;67:609–52. pmid:9759499
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Trowbridge JM, Gallo RL. Dermatan sulfate: new functions from an old glycosaminoglycan. Glycobiology. 2002;12(9):117R-25R. pmid:12213784
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Mizumoto S, Yamada S, Sugahara K. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins. Curr Opin Struct Biol. 2015;34:35–42. pmid:26164146
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Malmström A. Biosynthesis of dermatan sulphate. Loss of C-5 hydrogen during conversion of D-glucuronate to L-iduronate. Biochem J. 1981;198(3):669–75. pmid:7326032
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Pacheco B, Maccarana M, Malmström A. Dermatan 4-O-sulfotransferase 1 is pivotal in the formation of iduronic acid blocks in dermatan sulfate. Glycobiology. 2009;19(11):1197–203. pmid:19661164
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Mizumoto S, Yamada S. Histories of Dermatan Sulfate Epimerase and Dermatan 4-O-Sulfotransferase from Discovery of Their Enzymes and Genes to Musculocontractural Ehlers-Danlos Syndrome. Genes (Basel). 2023;14:2.
View Article
Google Scholar

[25] View Article

[26] Google Scholar

[ref8] 8. Malmström A, Fransson LA. Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues. J Biol Chem. 1975;250(9):3419–25.
View Article
Google Scholar

[28] View Article

[29] Google Scholar

[ref9] 9. Dündar M, Muller T, Zhang Q, Pan J, Steinmann B, Vodopiutz J, et al. Loss of dermatan-4-sulfotransferase 1 function results in adducted thumb-clubfoot syndrome. Am J Hum Genet. 2009;85(6):873–82.
View Article
Google Scholar

[31] View Article

[32] Google Scholar

[ref10] 10. Miyake N, Kosho T, Mizumoto S, Furuichi T, Hatamochi A, Nagashima Y, et al. Loss-of-function mutations of CHST14 in a new type of Ehlers-Danlos syndrome. Hum Mutat. 2010;31(8):966–74. pmid:20533528
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref11] 11. Müller T, Mizumoto S, Suresh I, Komatsu Y, Vodopiutz J, Dundar M, et al. Loss of dermatan sulfate epimerase (DSE) function results in musculocontractural Ehlers-Danlos syndrome. Hum Mol Genet. 2013;22(18):3761–72. pmid:23704329
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref12] 12. Malfait F, Francomano C, Byers P, Belmont J, Berglund B, Black J, et al. The 2017 international classification of the Ehlers-Danlos syndromes. Am J Med Genet C Semin Med Genet. 2017;175(1):8–26. pmid:28306229
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref13] 13. Mizumoto S, Kosho T, Hatamochi A, Honda T, Yamaguchi T, Okamoto N, et al. Defect in dermatan sulfate in urine of patients with Ehlers-Danlos syndrome caused by a CHST14/D4ST1 deficiency. Clin Biochem. 2017;50(12):670–7. pmid:28238810
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref14] 14. Minatogawa M, Hirose T, Mizumoto S, Yamaguchi T, Nagae C, Taki M, et al. Clinical and pathophysiological delineation of musculocontractural Ehlers-Danlos syndrome caused by dermatan sulfate epimerase deficiency (mcEDS-DSE): A detailed and comprehensive glycobiological and pathological investigation in a novel patient. Hum Mutat. 2022;43(12):1829–36. pmid:35842784
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref15] 15. Brady AF, Demirdas S, Fournel-Gigleux S, Ghali N, Giunta C, Kapferer-Seebacher I, et al. The Ehlers-Danlos syndromes, rare types. Am J Med Genet C Semin Med Genet. 2017;175(1):70–115.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref16] 16. Minatogawa M, Unzaki A, Morisaki H, Syx D, Sonoda T, Janecke AR, et al. Clinical and molecular features of 66 patients with musculocontractural Ehlers-Danlos syndrome caused by pathogenic variants in CHST14 (mcEDS-CHST14). J Med Genet. 2022;59(9):865–77. pmid:34815299
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref17] 17. Kobayashi T, Fujishima F, Tokodai K, Sato C, Kamei T, Miyake N, et al. Detailed Courses and Pathological Findings of Colonic Perforation without Diverticula in Sisters with Musculocontractural Ehlers-Danlos Syndrome Caused by Pathogenic Variant in CHST14 (mcEDS-CHST14). Genes. 2023;14(5).
View Article
Google Scholar

[61] View Article

[62] Google Scholar

[ref18] 18. Qian H, Zhou T, Zheng N, Lu Q, Han Y. Case report: Multiple gastrointestinal perforations in a rare musculocontractural Ehlers-Danlos syndrome with multiple organ dysfunction. Front Genet. 2022;13:846529. pmid:36046248
View Article
PubMed/NCBI
Google Scholar

[64] View Article

[65] PubMed/NCBI

[66] Google Scholar

[ref19] 19. Bian S, Akyüz N, Bernreuther C, Loers G, Laczynska E, Jakovcevski I, et al. Dermatan sulfotransferase Chst14/D4st1, but not chondroitin sulfotransferase Chst11/C4st1, regulates proliferation and neurogenesis of neural progenitor cells. J Cell Sci. 2011;124(Pt 23):4051–63. pmid:22159417
View Article
PubMed/NCBI
Google Scholar

[68] View Article

[69] PubMed/NCBI

[70] Google Scholar

[ref20] 20. Nitahara-Kasahara Y, Mizumoto S, Inoue YU, Saka S, Posadas-Herrera G, Nakamura-Takahashi A, et al. A new mouse model of Ehlers-Danlos syndrome generated using CRISPR/Cas9-mediated genomic editing. Dis Model Mech. 2021;14(12):dmm048963. pmid:34850861
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref21] 21. Akyüz N, Rost S, Mehanna A, Bian S, Loers G, Oezen I, et al. Dermatan 4-O-sulfotransferase1 ablation accelerates peripheral nerve regeneration. Exp Neurol. 2013;247:517–30.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref22] 22. Hirose T, Mizumoto S, Hashimoto A, Takahashi Y, Yoshizawa T, Nitahara-Kasahara Y, et al. Systematic investigation of the skin in Chst14-/- mice: A model for skin fragility in musculocontractural Ehlers-Danlos syndrome caused by CHST14 variants (mcEDS-CHST14). Glycobiology. 2021;31(2):137–50. pmid:32601684
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref23] 23. Yoshizawa T, Mizumoto S, Takahashi Y, Shimada S, Sugahara K, Nakayama J, et al. Vascular abnormalities in the placenta of Chst14-/- fetuses: implications in the pathophysiology of perinatal lethality of the murine model and vascular lesions in human CHST14/D4ST1 deficiency. Glycobiology. 2018;28(2):80–9. pmid:29206923
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref24] 24. Hirose T, Takahashi N, Tangkawattana P, Minaguchi J, Mizumoto S, Yamada S, et al. Structural alteration of glycosaminoglycan side chains and spatial disorganization of collagen networks in the skin of patients with mcEDS-CHST14. Biochim Biophys Acta Gen Subj. 2019;1863(3):623–31. pmid:30553867
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref25] 25. Tang T, Li L, Tang J, Li Y, Lin WY, Martin F, et al. A mouse knockout library for secreted and transmembrane proteins. Nat Biotechnol. 2010;28(7):749–55.
View Article
Google Scholar

[91] View Article

[92] Google Scholar

[ref26] 26. Shimada S, Yoshizawa T, Takahashi Y, Nitahara-Kasahara Y, Okada T, Nomura Y, et al. Backcrossing to an appropriate genetic background improves the birth rate of carbohydrate sulfotransferase 14 gene-deleted mice. Exp Anim. 2020;69(4):407–13. pmid:32522905
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref27] 27. Lackey AI, Chen T, Zhou YX, Bottasso Arias NM, Doran JM, Zacharisen SM, et al. Mechanisms underlying reduced weight gain in intestinal fatty acid-binding protein (IFABP) null mice. Am J Physiol Gastrointest Liver Physiol. 2020;318(3):G518–30. pmid:31905021
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref28] 28. Chassaing B, Aitken JD, Malleshappa M, Vijay-Kumar M. Dextran sulfate sodium (DSS)-induced colitis in mice. Curr Protoc Immunol. 2014;104:15.25.1-15.25.14. pmid:24510619
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref29] 29. Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993;69(2):238–49.
View Article
Google Scholar

[106] View Article

[107] Google Scholar

[ref30] 30. Ghia J-E, Blennerhassett P, Collins SM. Impaired parasympathetic function increases susceptibility to inflammatory bowel disease in a mouse model of depression. J Clin Invest. 2008;118(6):2209–18. pmid:18451995
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref31] 31. Hiura K, Maruyama T, Watanabe M, Nakano K, Okamura T, Sasaki H, et al. Mitotic spindle positioning protein (MISP) deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice. J Vet Med Sci. 2023;85(2):167–74. pmid:36596561
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref32] 32. Dieleman LA, Palmen MJ, Akol H, Bloemena E, Pena AS, Meuwissen SG, Van Rees EP. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 1998;114(3):385–91.
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref33] 33. Jialing L, Yangyang G, Jing Z, Xiaoyi T, Ping W, Liwei S, et al. Changes in serum inflammatory cytokine levels and intestinal flora in a self-healing dextran sodium sulfate-induced ulcerative colitis murine model. Life Sci. 2020;263:118587. pmid:33065145
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref34] 34. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. pmid:22743772
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref35] 35. Mizumoto S, Sugahara K. Glycosaminoglycan chain analysis and characterization (glycosylation/epimerization). Methods Mol Biol. 2012;836:99–115. pmid:22252630
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref36] 36. Eichele DD, Kharbanda KK. Dextran sodium sulfate colitis murine model: An indispensable tool for advancing our understanding of inflammatory bowel diseases pathogenesis. World J Gastroenterol. 2017;23(33):6016–29. pmid:28970718
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref37] 37. Hannesson HH, Hagner-McWhirter A, Tiedemann K, Lindahl U, Malmstrom A. Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism. Biochem J. 1996;313(Pt 2):589–96.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

[ref38] 38. Maccarana M, Olander B, Malmström J, Tiedemann K, Aebersold R, Lindahl U, et al. Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5-epimerase is identical to SART2. J Biol Chem. 2006;281(17):11560–8. pmid:16505484
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref39] 39. Fleischmajer R, Perlish JS, Burgeson RE, Shaikh-Bahai F, Timpl R. Type I and type III collagen interactions during fibrillogenesis. Ann N Y Acad Sci. 1990;580:161–75. pmid:2186689
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref40] 40. Stuart K, Panitch A. Characterization of gels composed of blends of collagen I, collagen III, and chondroitin sulfate. Biomacromolecules. 2009;10(1):25–31. pmid:19053290
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref41] 41. Keene DR, San Antonio JD, Mayne R, McQuillan DJ, Sarris G, Santoro SA, et al. Decorin binds near the C terminus of type I collagen. J Biol Chem. 2000;275(29):21801–4. pmid:10823816
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref42] 42. Maier F, Siri S, Santos S, Chen L, Feng B, Pierce DM. The heterogeneous morphology of networked collagen in distal colon and rectum of mice quantified via nonlinear microscopy. J Mech Behav Biomed Mater. 2021;113:104116. pmid:33049619
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref43] 43. Durcan C, Hossain M, Chagnon G, Perić D, Girard E. Mechanical experimentation of the gastrointestinal tract: a systematic review. Biomech Model Mechanobiol. 2024;23(1):23–59. pmid:37935880
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref44] 44. Feuerstein JD, Falchuk KR. Diverticulosis and Diverticulitis. Mayo Clin Proc. 2016;91(8):1094–104.
View Article
Google Scholar

[163] View Article

[164] Google Scholar

[ref45] 45. Tursi A. Current and Evolving Concepts on the Pathogenesis of Diverticular Disease. J Gastrointestin Liver Dis. 2019;28:225–35. pmid:31204408
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref46] 46. Wedel T, Barrenschee M, Lange C, Cossais F, Böttner M. Morphologic Basis for Developing Diverticular Disease, Diverticulitis, and Diverticular Bleeding. Viszeralmedizin. 2015;31(2):76–82. pmid:26989376
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref47] 47. Barbaro MR, Cremon C, Fuschi D, Marasco G, Palombo M, Stanghellini V, Barbara G. Pathophysiology of Diverticular Disease: From Diverticula Formation to Symptom Generation. Int J Mol Sci. 2022;23(12).
View Article
Google Scholar

[174] View Article

[175] Google Scholar

[ref48] 48. Patel B, Guo X, Noblet J, Chambers S, Kassab GS. Animal Models of Diverticulosis: Review and Recommendations. Dig Dis Sci. 2018;63(6):1409–18.
View Article
Google Scholar

[177] View Article

[178] Google Scholar

[ref49] 49. Zullo A. Medical hypothesis: speculating on the pathogenesis of acute diverticulitis. Ann Gastroenterol. 2018;31(6):747–9.
View Article
Google Scholar

[180] View Article

[181] Google Scholar

[ref50] 50. Piscopo N, Ellul P. Diverticular Disease: A Review on Pathophysiology and Recent Evidence. Ulster Med J. 2020;89(2):83–8. pmid:33093692
View Article
PubMed/NCBI
Google Scholar

[183] View Article

[184] PubMed/NCBI

[185] Google Scholar

[ref51] 51. Taylor KR, Rudisill JA, Gallo RL. Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity. J Biol Chem. 2005;280(7):5300–6. pmid:15563459
View Article
PubMed/NCBI
Google Scholar

[187] View Article

[188] PubMed/NCBI

[189] Google Scholar

[ref52] 52. Stevens C, Walz G, Singaram C, Lipman ML, Zanker B, Muggia A, et al. Tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 expression in inflammatory bowel disease. Dig Dis Sci. 1992;37(6):818–26.
View Article
Google Scholar

[191] View Article

[192] Google Scholar

[ref53] 53. Al-Sadi R, Boivin M, Ma T. Mechanism of cytokine modulation of epithelial tight junction barrier. Front Biosci (Landmark Ed). 2009;14(7):2765–78. pmid:19273235
View Article
PubMed/NCBI
Google Scholar

[194] View Article

[195] PubMed/NCBI

[196] Google Scholar

[ref54] 54. Biancheri P, Giuffrida P, Docena GH, MacDonald TT, Corazza GR, Di Sabatino A. The role of transforming growth factor (TGF)-β in modulating the immune response and fibrogenesis in the gut. Cytokine Growth Factor Rev. 2014;25(1):45–55. pmid:24332927
View Article
PubMed/NCBI
Google Scholar

[198] View Article

[199] PubMed/NCBI

[200] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Animal studies

Genotyping

Analysis of food intake, body weight, and fecal volume

Bead expulsion time

Intestinal transit time

Dextran sulfate sodium-induced colitis model

Sample collection

Strength and extensibility test

Pathological analysis

Immunofluorostaining of lymphocyte antigen-6 family member G (Ly-6G)

Transmission electron microscopy (TEM)

Scanning electron microscopy (SEM)

Western blot analysis

Quantitative analysis of DS and CS disaccharides

Real-time quantitative reverse transcription PCR

Statistical analysis

Results

DS is absent from the colon of Chst14-/- mice

Chst14-/- mice have shorter colons with increased contractility

Physiological colon function does not differ between Chst14+/+ and Chst14-/- mice

Collagen fibril deformation is induced by DS deficiency in the colon of Chst14-/- mice

Chst14-/- mice do not have more severe DSS-induced colitis compared with Chst14+/+ mice

Inflammatory response in DSS-induced colitis in DS deficiency mice

Discussion

Conclusions

Supporting information

S1 Fig. Gene expression levels related to colonic smooth muscle contraction.

S2 Fig. Colonic morphology, function, and collagen-related gene expression in middle-aged mice.

S3 Fig. Colonic morphology and collagen-related gene expression in female mice.

S4 Fig. Still images extracted from ultrasound videos showing the colons of Chst14+/+ and Chst14-/- mice.

S1 Movie. A representative ultrasound video of the colon from a Chst14+/+ mouse.

S2 Movie. A representative ultrasound video of the colon from a Chst14-/- mouse.

S1 Table. RT-qPCR primer sequences.

S2 Table. Relative mRNA levels.

S3 Table. Disaccharide composition of CS/DS, CS, and DS.

S4 Table. Physiological measurements.

S5 Table. Measured thicknesses of each layer.

S6 Table. Measured diameter of collagen fibrils.

S7 Table. Data of DSS colitis model.

S8 Table. Number of Ly-6G-positive cells.

S1 File. Raw images.

Acknowledgments

References

DS is absent from the colon of Chst14^-/- mice

Chst14^-/- mice have shorter colons with increased contractility

Physiological colon function does not differ between Chst14^+/+ and Chst14^-/- mice

Collagen fibril deformation is induced by DS deficiency in the colon of Chst14^-/- mice

Chst14^-/- mice do not have more severe DSS-induced colitis compared with Chst14^+/+ mice

S4 Fig. Still images extracted from ultrasound videos showing the colons of Chst14^+/+ and Chst14^-/- mice.

S1 Movie. A representative ultrasound video of the colon from a Chst14^+/+ mouse.

S2 Movie. A representative ultrasound video of the colon from a Chst14^-/- mouse.