DUSP26: Unveiling a critical molecular mediator and therapeutic target in developmental dysplasia of the hip‑associated secondary osteoarthritis

Introduction

Developmental dysplasia of the hip (DDH) is a congenital osteoarticular disorder, characterized by abnormal matching between the acetabulum and femoral head. Its severity can progress from mild acetabular dysplasia to subluxation or even complete dislocation (1). DDH disrupts the biomechanics of the hip joint, imposing an excessive load on the joint cartilage and giving rise to early-stage osteoarthritis (2,3). DDH is the predominant factor responsible for total hip arthroplasty among young people, accounting for ~21-29% of cases (2).

In the pathological progression of DDH, the degeneration of acetabular cartilage is a critical factor. As the tissue covering the outer surface of the acetabulum, its normal development is crucial for maintaining acetabular morphology (2). The acetabular cartilage is mainly composed of chondrocytes and the extracellular matrix (ECM) they secrete. Among the components of ECM, type II collagen (COL2A1) and proteoglycan aggrecan are the core elements (4). These core components not only offer a stable structural scaffold for chondrocytes but also help them withstand mechanical stress. Research has identified genetic factors (for example, GDF5 gene mutations) and mechanical stress (such as abnormal joint loading) as key causes of acetabular dysplasia (5), and has also revealed that pathways, such as Wnt/β-catenin, and deacetylase family proteins are involved in chondrocyte regulation (6-8). However, to the best of our knowledge, no studies have yet elucidated the specific molecular mechanisms underlying DDH-specific chondrocyte dysfunction (such as impaired ECM synthesis and excessive inflammatory responses), nor have they identified target molecules that can link regulatory pathways to DDH-related cartilage damage. This has markedly hindered the development of targeted therapies for DDH.

Dual-specificity phosphatases (DUSPs) are a subgroup of class I protein tyrosine phosphatases (PTPs) that uniquely dephosphorylate both tyrosine and serine/threonine residues, as well as lipid substrates in some cases. DUSP26, also known as MKP8, is classified as an atypical DUSP due to the absence of a conserved mitogen-activated protein kinase (MAPK)-binding domain, a structural feature that expands its substrate spectrum beyond classical MAPK targets (9). Functionally, DUSP26 negatively regulates the MAPK signaling cascade by directly dephosphorylating ERK and p38, thereby modulating key biological processes including cell proliferation, lipid accumulation and axon regeneration (10-12). In addition, it mediates distinct physiological or pathological outcomes (for example, cell-cell adhesiveness, retinal differentiation, inflammation and tumorigenesis) through dephosphorylating a range of non-MAPK molecules, including kinesin-associated protein 3 (13), Fas-associated protein with death domain (14), nerve growth factor receptor TrkA (15), fibroblast growth factor receptor 1 (15), tumor protein p53 (16) and transforming growth factor β-activated kinase 1 (TAK1) (17). Notably, DUSP26 exerts anti-inflammatory effects by dephosphorylating TAK1, which inhibits TAK1-dependent JNK activation, and consequently alleviates hepatic steatosis and metabolic disturbance (17). Knockdown of DUSP26 promotes diabetic renal fibrosis, manifested as the accumulation of type I collagen (COL1A1) (18). COL1A1 is known as a prominent hallmark of cartilage degeneration in osteoarthritis. Elevated COL1A1 expression alters the mechanical properties of the chondrocyte ECM, rendering it more rigid and thus impairing normal chondrocyte function (4). Based on these findings, it was hypothesized that DUSP26 might serve a critical role in the progression of cartilage degeneration and secondary osteoarthritis induced by DDH.

In the present study, a swaddling-induced DDH rat model was established and IL-1β-treated rat chondrocytes were employed to simulate an in vitro cartilage degeneration environment. Through proteomics analysis, the current study aimed to investigate whether DUSP26 was involved in DDH-related chondrocyte dysfunction and to clarify the molecular mechanism by which DUSP26 regulates DDH-associated chondrocytes. To the best of our knowledge, this is the first study to assess the role of DUSP26 in DDH. The findings may improve the understanding of the molecular mechanisms underlying DDH, and could provide a new target for developing targeted treatments for DDH.

Materials and methods

Animal DDH models

A total of three male and six female Wistar rats (age, 8-10 weeks; male weight, 280-320 g; female weight, 210-240 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and bred overnight in a 1:2 ratio to obtain 0-day-old neonatal pups (weight, 5-6 g). Wistar rats and neonatal rats were maintained in a specific pathogen-free facility, which was operated at 22±1°C temperature and 50±5% humidity, under a 12-h light/dark cycle with free access to food and water. A swaddling-induced DDH rat model was established, with the construction of this model strictly performed according to a previously reported protocol (19). Briefly, the 0-day-old neonatal rats (n=31) were randomly divided into the Control group (n=12; seven male and five female) and the DDH group (n=19; 10 male and nine female). The rats in the DDH group were subjected to straight-leg swaddling that restricted hip abduction and extension, as well as knee extension, with medical bandages for 10 postnatal days. The bandages for swaddling in the DDH group were changed every 2 days, and the rats were allowed to move freely for 4 h. Rats in the control group received no interventions. Subsequently, all rats were raised until they reached 4 weeks of age, anesthetized by isoflurane inhalation (3% for induction and 2% for maintenance), and euthanized via exsanguination. The hip joint was isolated for further analyses.

Histological examination

Before histological examination, the rat hip joints were macroscopically evaluated. Specifically, the hip joints were visually inspected with the naked eye to assess for obvious abnormalities, including dislocation, deformity and cartilage wear. Subsequently, the hip joints were fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and cut into 5-μm sections. Dewaxed sections were stained with hematoxylin for 5 min and eosin for 3 min, or Safranin O (SO) for 5 min and Fast Green (FG) for 1 min, all at room temperature, to evaluate the severity of cartilage lesions. Following dehydration with ethanol, permeabilization in xylene and mounting with neutral gum, images of the histological sections were captured under a light microscope at a magnification of ×40.

Genomic context and protein feature DUSP26

The genomic distribution and chromosomal localization of the DUSP26 gene were derived from NCBI (https://www.ncbi.nlm.nih.gov/gene). The protein structure of DUSP26-N was acquired from the Protein Data Bank with the accession code 5GTJ (https://www.rcsb.org/).

Immunohistochemical staining

Acetabular cartilage samples were meticulously harvested from rat hip joints with the aid of microsurgical tools under microscopic guidance. Subsequently, the acetabular cartilage was fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and sliced into 5-μm sections. The sections were deparaffinized, rehydrated through a descending ethanol series (95, 85 and 75%) and treated with boiling antigen retrieval solution for 10 min followed by incubation with 3% H2O2 for 15 min at room temperature. After blocking with 1% bovine serum albumin (BSA; cat. no. A602440-0050; Sangon Biotech Co., Ltd.) for 15 min at room temperature, the sections were incubated with primary antibodies overnight at 4°C, each diluted to a ratio of 1:200. These antibodies included those specific for DUSP26 (cat. no. bs-7910R; BIOSS), COL2A1 (cat. no. AF0135; Affinity Biosciences), histone deacetylase (HDAC)1 (cat. no. AF6433; Affinity Biosciences), phosphorylated (p)-HDAC1 (Ser421) (cat. no. PA5-36810; Thermo Fisher Scientific, Inc.), HDAC2 (cat. no. AF6470; Affinity Biosciences), p-HDAC2 (Ser394) (cat. no. bs-5389R; BIOSS), HDAC8 (cat. no. AF6481; Affinity Biosciences) and p-HDAC8 (Ser39) (cat. no. AF3481; Affinity Biosciences). Following an extensive washing process in PBS, the sections were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd.) at dilution of 1:100 in PBS at 37°C for 45 min followed by a staining with 3,3'-diaminobenzidine. The sections then underwent counterstaining with hematoxylin for 3 min at room temperature, dehydration with ethanol and xylene successively, and were finally mounted with neutral gum. To assess the expression levels of the target protein, Image-Pro Plus 6.0 (Media Cybernetics, Inc.) was used to assess integrated optical density (IOD) of the positively stained region. The mean optical density of the target protein in each image was calculated as IOD/stained area. Each sample was analyzed on a single slice by imaging three representative fields of view at ×400 magnification.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from rat acetabular cartilage using TRIpure Total RNA Extraction Reagent (cat. no. RP1001; BioTeke Corporation). cDNA was synthesized from 1 μg RNA using an All-in-One First-Strand SuperMix Kit according to the manufacturer's instructions (cat. no. MD80101; Guangzhou Magen Biotechnology Co., Ltd.). Subsequently, qPCR was performed in a 20-μl reaction mixture containing 1 μl cDNA, 0.5 μl SYBR green I (cat. no. SR4110; Beijing Solarbio Science & Technology Co., Ltd.), 10 μl 2X Fast Taq plus PCR Master Mix (cat. no. BL1014; Biosharp Life Sciences) and 0.5 μl each primer. qPCR was initiated with a 5-min denaturation step at 95°C, followed by 40 consecutive three-step cycles: Denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and extension at 72°C for 15 sec. Relative mRNA levels of target genes were calculated using the 2−ΔΔCq method (20) and normalized to the housekeeping gene β-actin. The primer sequences of target genes are listed in Table SI.

mRNA-sequencing (mRNA-seq) and biological analysis

mRNA-seq was performed by Novogene Co., Ltd. Briefly, total RNA from rat acetabular cartilage was extracted using TRNzol (cat. no. DP424; Tiangen Biotech Co., Ltd.). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (cat. no. N401-01/02; Vazyme Biotech Co., Ltd.). Standard RNA-seq library generation was performed using the Fast RNA-seq Lib Prep Kit V2 (cat. no. RK20306; ABclonal Biotech Co., Ltd.), according to the manufacturer's instructions, with an insert size of 250-300 bp, and subjected to paired-end 150 bp sequencing on the Illumina HWI-ST1276 instrument (Illumina, Inc.). Paired-end reads were filtered via: i) Removal of read pairs if either read contained adapter contamination; ii) exclusion of read pairs if uncertain bases constituted >10% of either read; and iii) elimination of read pairs if low-quality bases (Phred score <5) exceeded 50% of the total length in either read. Genes with significantly upregulated expression in the DDH group relative to the control group were identified using stringent criteria: Log2 fold change (FC) >4 and P.adj<0.01. Principal component analysis of significantly upregulated genes was performed using the DESeq2 (version 1.46.0) (21) and ggplot2 (version 3.5.1; https://ggplot2.tidyverse.org) in R package (version 4.4.2; https://www.r-project.org). A heatmap of the significantly upregulated genes was constructed using pheatmap (version 1.0.12; https://cran.r-project.org/package=pheatmap) in R package (version 4.4.2). For Gene Ontology (GO) annotations, the significantly upregulated genes were analyzed using the R packages ggplot2 and patchwork (https://patchwork.data-imaginist.com).

Primary culture of chondrocytes

For cartilage collection, another cohort of 75 0-day-old neonatal Wistar pups (weight, 5-6 g; 40 male and 35 female rats; housed as aforementioned) was raised to 5 days of age; subsequently, they were anesthetized with isoflurane (3% induction, 2% maintenance via inhalation) and euthanized by exsanguination. The tibial and femoral cartilages were dissected from 5-day-old rats as previously described (22). After washing in PBS, the cartilage pieces were digested with 3% (w/v) collagenase II (cat. no. BS164; Biosharp Life Sciences) twice for 45 min at 37°C each time. The primary chondrocytes (P0) were cultured in DMEM/F12 (cat. no. BL305A; Biosharp Life Sciences) containing 10% fetal bovine serum (cat. no. 11011-8611; Beijing Solarbio Science & Technology Co., Ltd.) in a humidified incubator at 37°C with 5% CO2. Primary chondrocytes were cultured for 3 days until they reached 90% confluence prior to passage. Cells from the P0 and P1 generations in the logarithmic growth phase were selected for subsequent experiments. Cell contamination was routinely excluded by microscopic examination of cell morphology and culture medium condition.

Immunofluorescence assay

Chondrocytes were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized using 0.1% Triton X-100 for 30 min at room temperature and subsequently blocked with 1% BSA for 15 min at room temperature. Cells were then incubated with anti-COL2A1 antibody (1:100; cat. no. AF0135; Affinity Biosciences) overnight at 4°C. After an extensive washing process in PBS, the cells were incubated with Cy3-labeled goat anti-rabbit IgG secondary antibody (1:200; cat. no. SA00009-2; Wuhan Sanying Biotechnology) for 1 h at room temperature. After staining the nuclei with DAPI at room temperature for 5 min and mounting with anti-fluorescence quencher, immunofluorescence images were captured under a fluorescence microscope at a magnification of ×400.

Chondrocytes treated with interleukin (IL)-1β

To assess the functional integrity of chondrocytes, the P0-P1 primary chondrocytes were treated with the pro-inflammatory cytokine IL-1β (10 ng/ml; cat. no. HY-P7097; MedChemExpress) with or without the HDAC inhibitor trichostatin A (TSA; 300 nM; cat. no. T129665; Shanghai Aladdin Biochemical Technology Co., Ltd.) for 24 h at room temperature (22,23).

Construction of adenovirus vectors

For knockdown of DUSP26, two short hairpin RNAs (shRNAs) specifically targeting rat DUSP26 mRNA were inserted into the BglII/SalI sites of the pShuttle-CMV-H1 adenoviral vector (cat. no. BR009; Hunan Fenghui Biotechnology Co., Ltd.) (target sequence-1, 5'-ACTCGCCAGCCTTTGACATGA-3'; target sequence-2, 5'-CCTCATGCTGTACCACCACTT-3'). For knockdown of HDAC1/2/8, shRNAs targeting rat HDAC1/2/8 mRNA were inserted into the BglII/SalI sites of the pShuttle-CMV-H1 adenoviral vector. The specific target sequences were as follows: HDAC1, 5'-GCTGCTCAACTATGGTCTCTA-3'; HDAC2, 5'-CCCAATGAGTTGCCATATAAT-3'; and HDAC8, 5'-CTACAGTGTCAATGTGCCCAT-3'. A non-specific shRNA (target sequence, 5'-TTCTCCGAACGTGTCACGT-3') was inserted into the pShuttle-CMV-H1 adenoviral vector to generate the adenoviral negative control (Ad-shNC). For overexpression of DUSP26, the rat DUSP26 CDS fragment (NM_001012352) was inserted into the KpnI/NotI sites of the pShuttle-CMV adenoviral vector. An empty pShuttle-CMV adenoviral vector served as a control (Ad-EP). Once constructed, the shuttle vector containing shRNAs or the rat DUSP26 CDS fragment was linearized with FastDigest PacI (cat. no. FD2204; Thermo Fisher Scientific, Inc.) and co-transformed into Escherichia coli BJ5183 cells (Beijing Huayueyang Biological Technology Co., Ltd.) with pAdEasy-1 backbone plasmid (cat. no. 16400; Addgene, Inc.) following the manufacturer's recommended ratio (1:1, by mass) for homologous recombination. The resulting recombinant adenoviral plasmids (25 μg) were then transfected into 293A packaging cells (iCell Bioscience, Inc.) using a high-efficiency transfection reagent (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at room temperature. Cells were cultured for 10-14 days until the cytopathic effect reached >50%. Cells were then harvested by repeated freeze-thaw and filtered through a 0.45-μm filter to obtain the first-generation virus. Separate second-generation viral stocks were generated for each recombinant adenoviral vector via one additional round of amplification: Specifically, the two DUSP26 knockdown vectors (Ad-shDUSP26-1, Ad-shDUSP26-2), three HDAC knockdown vectors (Ad-shHDAC1, Ad-shHDAC2, Ad-shHDAC8), one DUSP26 overexpression vector (Ad-DUSP26), and two control vectors (Ad-shNC, Ad-EP) each had corresponding second-generation viral stocks. These second-generation viral stocks were used to infect chondrocytes at multiplicity of infection values of 100. After a 24-h incubation period, the chondrocytes were subjected to IL-1β stimulation.

ELISA

Tissues were weighed, homogenized with normal saline (9X volume) on ice, centrifuged at 663 × g for 10 min at 4°C and the supernatants were collected. Cell supernatants from the primary chondrocytes were collected by centrifugation at 300 × g for 10 min at 4°C. Protein concentrations were measured using a BCA assay kit (cat. no. PK10026; Wuhan Sanying Biotechnology), and the levels of TNF-α and IL-6 in the tissue homogenates and cell supernatants were measured using the Rat TNF-α ELISA Kit (cat. no. EK382; Multi Sciences Biotech) and Rat IL-6 ELISA Kit (cat. no. EK306; Multi Sciences Biotech), according to the manufacturers' instructions.

Western blotting

Proteins were extracted from the primary chondrocytes on ice using a RIPA lysis buffer (cat. no. PR20001; Wuhan Sanying Biotechnology) containing 1% protease inhibitor (cat. no. PR20032; Wuhan Sanying Biotechnology) and phosphatase inhibitor (cat. no. PR20015; Wuhan Sanying Biotechnology) for 30 min. The supernatant fraction was collected by centrifugation at 10,000 × g for 3 min at 4°C, and the protein concentration was determined using a BCA assay kit. Subsequently, 15-30 μg proteins (loading volume of 15 μl per lane) were separated by SDS polyacrylamide gel electrophoresis on 10 and 12% gels and transferred onto a PVDF membrane. The membrane was then incubated in a blocking solution (cat. no. PR20011; Wuhan Sanying Biotechnology) for 2 h at room temperature, after which, it was probed with primary antibodies at 4°C overnight. These antibodies included anti-DUSP26 (1:1,000; cat. no. GTX109283; GeneTex, Inc.), anti-COL2A1 (1:500; cat. no. AF0135; Affinity Biosciences), anti-COL1A1 (1:500; cat. no. AF7001; Affinity Biosciences), anti-TNF-α (1:500; cat. no. AF7014; Affinity Biosciences), anti-HDAC1 (1:500; cat. no. AF6433; Affinity Biosciences), anti-p-HDAC1 (Ser421) (1:500; cat. no. PA5-36810; Thermo Fisher Scientific, Inc.), anti-HDAC2 (1:500; cat. no. AF6470; Affinity Biosciences), anti-p-HDAC2 (Ser394) (1:500; cat. no. bs-5389R; BIOSS), anti-HDAC8 (1:500; cat. no. AF6481; Affinity Biosciences), anti-p-HDAC8 (Ser39) (1:500; cat. no. AF3481; Affinity Biosciences), anti-β-catenin (1:1,000; cat. no. AF6266; Affinity Biosciences) and anti-β-actin (1:20,000; cat. no. 66009-1-Ig; Wuhan Sanying Biotechnology). Subsequently, the membrane was incubated with HRP-conjugated goat anti-rabbit (cat. no. SA00001-2; Wuhan Sanying Biotechnology) or anti-mouse (cat. no. SA00001-1; Wuhan Sanying Biotechnology) secondary antibodies at 1:10,000 dilution at 37°C for 40 min. The protein bands were visualized using an ultrasensitive enhanced chemiluminescent detection kit (cat. no. PK10003; Wuhan Sanying Biotechnology) according to the manufacturer's instructions and were analyzed by Tanon 5200 Image Analysis software (Tanon Science and Technology Co., Ltd.).

Co-immunoprecipitation (Co-IP)

The primary chondrocytes were infected with a DUSP26-overexpressing adenovirus followed by treatment with IL-1β. The Co-IP assay was performed using a Pierce Co-IP Kit (cat. no. 26149; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, proteins were extracted from the primary chondrocytes on ice using an IP lysis buffer containing 1% protease inhibitor (cat. no. PR20032; Wuhan Sanying Biotechnology) and phosphatase inhibitor (cat. no. PR20015; Wuhan Sanying Biotechnology) for 30 min. The supernatant fraction of cell lysates was collected by centrifugation at 10,000 × g for 3 min at 4°C, and the protein concentration was determined using a BCA assay kit. An aliquot of the cell lysate supernatant (500 μg) was reserved as the Input group prior to IP. For each Co-IP reaction, 2 μl anti-DUSP26 antibody (cat. no. GTX109283; GeneTex, Inc.) or anti-rabbit IgG isotype control (cat. no. 30000-0-AP; Wuhan Sanying Biotechnology) was pre-conjugated to 20 μl aldehyde-activated agarose beads in 200 μl 1X coupling buffer and 3 μl sodium cyanoborohydride for 2 h at room temperature with gentle rotation. After washing twice with IP lysis buffer, the antibody-conjugated beads were incubated with 500 μg cell lysate for 2 h at room temperature with gentle rotation, and the bound protein complexes were eluted with elution buffer for 5 min at room temperature. The eluates were collected by centrifugation at 1,000 × g for 1 min at 4°C and subjected to western blot analysis.

Mass spectrometry (MS)

For protein identification by MS analysis, protein lysates were obtained from primary chondrocytes infected with Ad-DUSP26and treated with IL-1β as aforementioned, and were reduced with 10 mM dithiothreitol at 37°C for 60 min, alkylated with 55 mM iodoacetamide at room temperature in the dark for 30 min, diluted with 50 mM ammonium bicarbonate for 10 min and subsequently digested with sequencing-grade trypsin overnight at 37°C. The resulting peptides were desalted using C18 columns, which were activated by 100% acetonitrile and equilibrated using 0.1% formic acid before loading samples. Subsequently, the columns were washed using 0.1% formic acid, and peptide fragments were eluted using 70% acetonitrile and lyophilized. Next, the lyophilized powder was dissolved in 10 μl mobile phase A (0.1% formic acid in water) and then centrifuged at 14,000 × g for 20 min at 4°C. Subsequently, the supernatants (4 μl) were injected into the UltiMate 3000 system (Thermo Fisher Scientific, Inc.) equipped with a column (inner diameter, 150 μm) packed with ReproSil-Pur C18-AQ 1.5 μm silica beads. Next, gradient elution was carried out using mobile phase B (0.1% formic acid in 80% acetonitrile) at a flow rate of 300 nl/min. The elution gradients were as follows: 8% B for 5 min, 12% B for 5 min, 30% B for 35 min, 40% B for 44 min, 95% B for 45 min and 95% B for 60 min. Following HPLC fractionation, the eluted peptide fractions were introduced into the Q Exactive HF-X mass spectrometer via a Nanospray Flex™ (NSI) ion source operating in positive ion mode, with a spray voltage of 2.2 kV and a capillary temperature of 320°C. For MS detection, a data-dependent acquisition mode was employed: The full-scan MS1 spectra (m/z range: 350-1,500) were acquired at a resolution of 120,000 (200 m/z), with an automatic gain control (AGC) target of 3×106 and a maximum injection time of 80 msec. The top 40 most abundant precursor ions were subsequently selected for MS2 fragmentation via higher-energy collisional dissociation with a normalized collision energy of 27%. The MS2 spectra were acquired at a resolution of 15,000 (200 m/z), with an AGC target of 5×104 and a maximum injection time of 45 msec. Data acquisition was performed by Beijing Qinglian Biotechnology Co., Ltd. and analyzed using the Proteome Discoverer 2.4 software (Thermo Fisher Scientific, Inc.), with searching against the Rattus_norvegicus_UP000002494_uniprot database (https://www.uniprot.org/proteomes/UP000002494). IgG was used to exclude non-specific interactors of DUSP26 in chondrocytes under IL-1β stimulation.

Integrative analysis of DUSP26-associated genes in chondrocyte degeneration

DUSP26-specific interactors were identified by IP/MS analysis. Non-differentially expressed genes were selected from mRNA-seq data using the following screening criteria: −1<log2FC<1 and P≥0.05. GeneCards (https://www.genecards.org/) was used to retrieve genes associated with 'osteoarthritis', 'cartilage degradation' and 'inflammation'. The jvenn online tool (https://jvenn.toulouse.inrae.fr/app/index.html) was employed to visualize the intersection of gene sets using Venn diagrams. Finally, candidate genes related to 'osteoarthritis', 'chondrocytes' and 'phosphorylation' were identified through PubMed literature search engines (https://pubmed.ncbi.nlm.nih.gov/).

Statistical analysis

Analyses were performed using GraphPad Prism 8.0.2 (Dotmatics). The normal distribution of the data was determined using the Shapiro-Wilk test. One-way analysis of variance followed by Tukey test was employed for comparisons among multiple groups. Unpaired Student's t-test was used for comparisons between two groups. All data are presented as the mean ± SD. All quantitative experiments were conducted with at least three independent biological replicates, each including technical triplicates. P<0.05 was considered to indicate a statistically significant difference.

Results

Acetabular cartilage degeneration occurs in rats with swaddling-induced DDH

Neonatal rats were subjected to swaddling for 10 days. A total of 4 weeks later, the hip joints of the rats were macroscopically evaluated. Compared with the control rats, the rats that had undergone straight-leg swaddling exhibited shallower acetabular cavities, and their femoral heads were small and oval-shaped (Fig. 1Aa). Histologically, femoral head dislocation and acetabular labral tears occurred in the rats undergoing straight-leg swaddling (Fig. 1Ab), and the SO/FG staining intensity on the acetabular surface was weakened (Fig. 1Ac). Immunohistochemical staining of the acetabular cartilages from the 4-week-old rats showed a decreased expression of COL2A1, a major collagen synthesized by chondrocytes (Fig. 1B and E). Gene expression analysis further confirmed that the expression of COL2A1 was downregulated in the acetabular cartilage of the 4-week-old rats with DDH, whereas the expression levels of IL-1β and IL-6 were upregulated (Fig. 1C). Furthermore, ELISA showed a higher level of IL-6 in the acetabular cartilage of the 4-week-old rats with DDH (Fig. 1D). These results demonstrated that acetabular collagen degradation and inflammatory responses occurred in the rats with DDH induced by swaddling.

Figure 1 Significant Ups genes in the acetabular cartilage of the rats with DDH, as determined by mRNA-seq analysis. (A) Neonatal rats were subjected to straight-leg swaddling for 10 days. A total of 4 weeks later, (a) morphology, (b) H&E staining and (c) SO/FG staining were assessed in the hip joints of the rats. (B) Immunohistochemical staining and (E) semi-quantitative analysis of COL2A1 in the acetabular cartilage of the 4-week-old control rats and rats with DDH. (C) Relative gene expression levels of COL2A1, IL-1β and IL-6 in the acetabular cartilage of the 4-week-old control rats and rats with DDH, as determined by reverse transcription-quantitative PCR. (D) Levels of IL-6 in the supernatants of acetabular cartilage from the 4-week-old rats with DDH, as determined by ELISA. (F) Significant Ups in the acetabular cartilage of rats with DDH were identified by mRNA-seq analysis with the screening criteria of log2FC >4 and P.adj<0.01. Principal component analysis results and a heatmap of these significant Ups are shown. (G) GO enrichment analysis of significant Ups in the acetabular cartilage of the rats with DDH. Data are presented as the mean ± SD. ***P<0.001. (C-E) Representative results of four independent experiments are shown. Ups, upregulated genes; BP, biological process; CC, cellular component; COL2A1, type II collagen; DDH, developmental dysplasia of the hip; FC, fold change; GO, Gene Ontology; H&E, hematoxylin and eosin; IHC, immunohistochemistry; IL, interleukin; IOD, integrated optical density; MF, molecular function; mRNA-seq, mRNA sequencing; SO/FG, Safranin O/Fast Green.

Significantly upregulated genes in the acetabular cartilage of rats with DDH, as determined by mRNA-seq

To identify the key factor in DDH induced by swaddling, mRNA-seq analysis of the acetabular cartilage was conducted. With the screening criteria of log2FC >4 and P.adj<0.01, 117 markedly upregulated genes (subsequently referred to as 'Ups') in the acetabular cartilage of the rats with DDH were obtained (Fig. 1F). The expression trends of IL-1β and COL2A1 detected by RT-qPCR were fully consistent with the sequencing data (Fig. 1C), confirming the accuracy of the differentially expressed gene screening. Subsequently, the GO-biological process enrichment analysis showed that the Ups were mainly related to 'muscle cell differentiation' and 'muscle cell development'. The GO-cellular component enrichment analysis indicated that the Ups were mainly associated with 'myofibril' and 'sarcomere'. The GO-molecular function enrichment analysis revealed that the Ups were mainly involved in aspects such as 'structural constituent of muscle' and 'actin binding' (Fig. 1G).

DUSP26 is highly expressed in the acetabular cartilage of rats with DDH

DDH is a common abnormality leading to osteoarthritis (24). Therefore, the present study aimed to identify a novel gene that may be involved in osteoarthritis secondary to dysplasia of the hip. Genes related to cartilage degradation and osteoarthritis were searched using the GeneCards database. Venn interaction analysis revealed that 38 Ups were associated with cartilage degradation, with known osteoarthritic genes excluded (Fig. 2A). Among them, the DUSP26 gene, located on chromosome 8p12, has an active PTP-loop conformation (Fig. 2B and C). DUSP26 regulates intracellular signaling pathways by dephosphorylating the tyrosine and serine/threonine residues of proteins (9). Immunohistochemical staining showed higher DUSP26 expression in the acetabular cartilage of 4-week-old rats with DDH compared with that in the control group (Fig. 2D). Moreover, the mRNA levels of DUSP26 were upregulated in the cartilage of the acetabulum from 4-week-old rats with DDH (Fig. 2E).

Figure 2 DUSP26 is highly expressed in the acetabular cartilages of rats with DDH. (A) Three-set Venn diagrams of significant Ups identified by mRNA-seq analysis, and osteoarthritis- and cartilage degradation-related genes retrieved from the GeneCards database. (B) Genomic context of the DUSP26 gene in chromosome 8p12 derived from NCBI (https://www.ncbi.nlm.nih.gov/gene). (C) PTP-loop conformation of DUSP26-N. Protein Data Bank code: 5GTJ (https://www.rcsb.org/). (D) Immunohistochemical staining for DUSP26 in the acetabular cartilage of the 4-week-old control rats and rats with DDH. (E) Relative mRNA expression levels of DUSP26 in the acetabular cartilage of the 4-week-old control rats and rats with DDH, as determined by reverse transcription-quantitative PCR. Data are presented as the mean ± SD. ***P<0.001. (D and E) Representative results of four independent experiments are shown. DDH, developmental dysplasia of the hip; DUSP26, dual-specificity phosphatase 26; Ups, upregulated genes; IHC, immunohistochemistry; IOD, integrated optical density; mRNA-seq, mRNA sequencing; PTP, protein tyrosine phosphatase.

DUSP26 expression is upregulated in IL-1β-induced chondrocytes

To determine the effects of DUSP26 on chondrocyte degradation, primary rat chondrocytes were extracted and identified as chondrocytes by immunofluorescence staining of COL2A1 (Fig. S1). Subsequently, the chondrocytes were subjected to IL-1β stimulation with the intention of mimicking chondrocyte injury in vitro. Notably, the protein and mRNA expression levels of DUSP26 in chondrocytes induced by IL-1β were significantly increased (Fig. 3A). Subsequently, rat DUSP26 overexpression and knockdown adenoviruses were constructed and used to infect chondrocytes. After 24 h, the protein and mRNA expression levels of DUSP26 in the chondrocytes were effectively modulated (Fig. 3B and C).

Figure 3 DUSP26 expression is upregulated in IL-1β-induced chondrocytes. (A) Schematic diagram showing the experimental protocol in vitro. Primary chondrocytes isolated from 5-day-old rats were subjected to treatment with 10 ng/ml IL-1β. The protein and mRNA expression levels of DUSP26 in chondrocytes were detected by RT-qPCR and western blotting. (B) Rat DUSP26 overexpression adenovirus was constructed and used to infect chondrocytes. After 24 h, the protein and mRNA expression levels of DUSP26 in the chondrocytes were detected by RT-qPCR and western blotting. (C) Two adenovirus-mediated shRNAs against rat DUSP26 were constructed and used to infect chondrocytes. After 24 h, the protein and mRNA expression levels of DUSP26 in the chondrocytes were assessed by RT-qPCR and western blotting. Data are presented as the mean ± SD. **P<0.01 and ***P<0.001. Representative results of three independent experiments are shown. Ad-EP, empty adenoviral vector; DUSP26, dual-specificity phosphatase 26; IL, interleukin; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin.

DUSP26 overexpression alleviates IL-1β-induced chondrocyte injury

An inflammatory environment in vitro can activate catabolic processes of chondrocytes and exacerbate cartilage erosion (4). As demonstrated in Fig. 4A, IL-1β induced chondrocyte degeneration, as manifested as enhanced COL1A1 expression and reduced COL2A1 expression. Overexpression of DUSP26 prevented upregulation of COL1A1 and downregulation of COL2A1 caused by IL-1β (Fig. 4A). Furthermore, overexpression of DUSP26 lowered TNF-α and IL-6 levels in the chondrocytes under IL-1β stimulation (Fig. 4B and C). In line with this, knockdown of DUSP26 aggravated IL-1β-induced chondrocyte damage, as evidenced by increased expression of catabolic markers (COL1A1, TNF-α and IL-6) and decreased expression of the anabolic marker COL2A1 (Fig. 4D-F).

Figure 4 DUSP26 overexpression alleviates IL-1β-induced chondrocyte injury. Rat DUSP26 overexpression adenovirus was constructed and used to infect chondrocytes. After 24 h, the chondrocytes were subjected to 10 ng/ml IL-1β. (A) Expression levels of COL2A1 and COL1A1 in the chondrocytes were detected by RT-qPCR and western blotting. (B) Relative mRNA expression levels of TNF-α and IL-6 in the chondrocytes were determined by RT-qPCR. (C) Levels of TNF-α and IL-6 in the supernatants of the chondrocytes were determined by ELISA. Two adenovirus-mediated shRNAs against rat DUSP26 were constructed and used to infect chondrocytes. After 24 h, the chondrocytes were subjected to 10 ng/ml IL-1β. (D) Expression levels of COL2A1 and COL1A1 in the chondrocytes were evaluated by RT-qPCR and western blotting. (E) Relative mRNA expression levels of TNF-α and IL-6 in the chondrocytes were determined by RT-qPCR. (F) Levels of TNF-α and IL-6 in the supernatants of the chondrocytes were detected by ELISA. Data are presented as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001. Representative results of three independent experiments are shown. COL1A1, type I collagen; COL2A1, type II collagen; EP, empty adenoviral vector; DUSP26, dual-specificity phosphatase 26; IL, interleukin; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; sh, short hairpin.

HDAC1, HDAC2 and HDAC8 are DUSP26-interacting proteins in chondrocytes

Target proteins that bound to DUSP26 and whose activities were affected by the phosphatase DUSP26 were screened for. Firstly, 4,234 of proteins that specifically bound to DUSP26 in chondrocytes under IL-1β stimulation were harvested after the IP/MS analysis (Fig. 5A). Combining with the previous mRNA-seq results, 3,466 DUSP26-specific interactors with non-differential expression were obtained (Fig. 5B, two-set Venn diagram). Subsequently, the four-set Venn diagram revealed 538 DUSP26-specific interactors related to 'osteoarthritis', 'cartilage degradation' and 'inflammation' (Fig. 5B). Among them, activation of three Class I HDACs (HDAC1, HDAC2 and HDAC8) serves an important role in cartilage degeneration and osteoarthritis, as determined using search engines such as PubMed (Fig. 5B) (7,8). Therefore, the current study explored whether these three HDACs were involved in the progression of DUSP26-mediated osteoarthritis secondary to DDH. Increased HDAC1/2/8 total protein, p-HDAC1Ser421, p-HDAC2Ser394 and p-HDAC8Ser39 levels were observed in the acetabular cartilage of the rats with DDH in vivo (Figs. 5C and S2). In chondrocytes treated with IL-1β, total expression and phosphorylation levels of HDAC1/2/8 were also enhanced (Fig. 5D). Co-IP experiments confirmed that DUSP26 bound to HDAC1/2/8 in IL-1β-stimulated chondrocytes (Fig. 5E).

Figure 5 HDAC1, HDAC2 and HDAC8 are DUSP26-interacting proteins in chondrocytes. (A) IP/MS analysis in DUSP26-overexpressing chondrocytes under IL-1β stimulation. (B) Flow chart of target protein screening. (C) Immunohistochemical staining of HDAC1, HDAC2 and HDAC8 in the acetabular cartilage of the 4-week-old control rats and rats with DDH. (D) Protein expression levels of HDAC1, p-HDAC1, HDAC2, p-HDAC2, HDAC8 and p-HDAC8 in the chondrocytes by western blotting. (E) Co-IP experiments with DUSP26 protein from extracts of IL-1β-treated chondrocytes followed by western blotting with indicated antibodies. DEG, differentially expressed gene; DUSP26, dual-specificity phosphatase 26; HDAC, histone deacetylase; IB, immunoblot; IL, interleukin; IP, immunoprecipitation; LC-MS/MS, liquid chromatography-tandem MS; mRNA-seq, mRNA sequencing; MS, mass spectrometry; p-, phosphorylated.

Inactivation of HDAC1/2/8 inhibits DUSP26 silencing-triggered cartilage degeneration

Under IL-1β stimulation, overexpression of DUSP26 decreased the phosphorylation levels of HDAC1, HDAC2 and HDAC8 in chondrocytes, but did not affect the abundance of total proteins (Fig. 6A). By contrast, silencing of DUSP26 increased their phosphorylation levels. Adenovirus-mediated shRNAs against HDAC1, HDAC2 and HDAC8 were constructed and used to infect chondrocytes. After 24 h, the expression levels of HDAC1, HDAC2 and HDAC8 mRNA and protein in chondrocytes were effectively knocked down, respectively (Figs. 6B and S3). After the chondrocytes were co-infected with the HDAC1/2/8 interference adenoviruses and the DUSP26 interference adenovirus, the mRNA and protein expression levels of the catabolic marker COL1A1 and the inflammatory marker TNF-α were decreased relative to the Ad-shDUSP26 group (Fig. 6C and E). Furthermore, intervention with TSA, an inhibitor of HDAC activity, effectively reversed DUSP26 silencing-induced COL1A1 and TNF-α upregulation (Fig. 6D and F). These findings suggest that the DUSP26-HDAC1/2/8 phosphorylation regulatory axis may act as a key mechanism underlying the inhibition of cartilage degeneration. In addition, high β-catenin expression was observed in the acetabular cartilage of rats with DDH (Fig. S4). Therefore, DUSP26 may serve a potential synergistic protective role via the β-catenin pathway.

Figure 6 Inactivation of HDAC1/2/8 inhibits DUSP26 silencing-triggered cartilage degeneration. (A) Total protein and phosphorylation levels of HDAC1, HDAC2 and HDAC8 in IL-1β-treated chondrocytes determined by western blotting. (B) Relative mRNA expression levels of HDAC1, HDAC2 and HDAC8 in chondrocytes, as determined by reverse transcription-quantitative PCR. (C) Relative mRNA expression levels of COL1A1 and TNF-α in IL-1β-treated chondrocytes after knocking down HDAC1/2/8. (D) Relative mRNA expression levels of COL1A1 and TNF-α in IL-1β-treated chondrocytes after intervention with the HDAC inhibitor TSA. (E) Protein levels of COL1A1 and TNF-α in IL-1β-treated chondrocytes after knocking down HDAC1/2/8. (F) Protein levels of COL1A1 and TNF-α in IL-1β-treated chondrocytes after intervention with the HDAC inhibitor TSA. Data are presented as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001. (B-D) Representative results of three independent experiments are shown. COL1A1, type I collagen; DUSP26, dual-specificity phosphatase 26; HDAC, histone deacetylase; IL, interleukin; NC, negative control; ns, no significance; sh, short hairpin; TSA, trichostatin A.

Discussion

Secondary osteoarthritis is caused by known factors, with DDH being a prominent example. Patients with DDH-osteoarthritis undergo total hip replacement at a markedly younger age than those with primary hip osteoarthritis (25). Acetabular dysplasia serves as a major instigator of early-stage hip osteoarthritis. Clinical evidence has indicated that individuals afflicted with acetabular dysplasia face a 1.1-10.2 higher risk of developing osteoarthritis than those without this abnormality (26). Compared with the normal acetabulum, acetabular dysplasia results in a diminished weight-bearing surface, which in turn augments contact stress, precipitating acetabular labral tears and/or cartilage damage. The present study aimed to identify the molecular targets implicated in osteoarthritis triggered by acetabular dysplasia, with the hope of providing preventive strategies for DDH-related joint degeneration. In the present study, rats with swaddling-induced DDH exhibited pronounced acetabular cartilage degeneration and inflammatory responses. Notably, DUSP26 exhibited a markedly elevated expression level in the acetabular cartilage of rats with DDH. Given the anti-inflammatory and anti-collagen degradation effects of DUSP26, high expression of DUSP26 may prevent the development of secondary osteoarthritis by DDH.

In osteoarthritis, articular chondrocytes invariably undergo hypertrophy, and the cartilage matrix suffers damage. The phenotype of these chondrocytes transitions into hypertrophic and fibrochondrocytic states, accompanied by a notable upsurge in the production of COL1A1. In parallel, the ECM synthesis is impeded as a result of chondrocyte dedifferentiation and apoptosis (27). HDACs are an important class of enzymes in the process of histone modification, which catalyze and regulate histone deacetylation. HDACs are categorized into four classes based on their homology with yeast proteins. Class I HDACs typically exert an impact on chromatin structure and gene transcription (28). Studies have shown that members of class I HDACs are important in human cartilage biology and the development of osteoarthritis (7,8,29). Specifically, HDAC1 and HDAC2 downregulate the expression of cartilage-specific proteins, such as COL2A1 and aggrecan, by binding to the transcriptional repressor Snail (7). The enhanced expression of HDAC8 prevents chondrogenic differentiation and reduces cartilage matrix synthesis (8). Notably, phosphorylation of HDAC1 at Ser421/423, HDAC2 at Ser394 and HDAC8 at Ser39 enhances the deacetylation function of their respective proteins and subsequently affects gene expression. Conversely, the HDAC deacetylase inhibitor TSA has proven effective in suppressing chondrocyte degradation (29), further validating HDACs as key drivers of osteoarthritis.

In the present study, the results suggested that DUSP26 may inhibit cartilage degeneration by directly suppressing the phosphorylation of HDAC1/2/8. Overexpression of DUSP26 inhibited chondrocyte ECM degradation instigated by IL-1β, manifested by an increase in the synthetic marker COL2A1, and a decrease in the catabolic markers COL1A1, TNF-α and IL-6. Either knocking down HDAC1, HDAC2 and HDAC8, or using the deacetylase inhibitor TSA could reverse the chondrocyte catabolism triggered by the knockdown of DUSP26. The present study revealed that the DUSP26-HDAC1/2/8 phosphorylation regulatory axis may act as a key mechanism underlying the inhibition of cartilage degeneration. It has been indicated that DUSP26 serves a pivotal role in promoting the translocation of β-catenin to intercellular junction sites. It achieves this by binding to KAP3 and subsequently inhibiting its phosphorylation (13). Such a mechanism suggests that DUSP26 likely wields influence over the distribution dynamics of β-catenin, both within and outside the cell nucleus. Notably, activation of β-catenin serves to accelerate the degradation of the chondrocyte ECM, thereby fueling the progression of osteoarthritis (30,31). In the present study, high β-catenin expression was observed in the acetabular cartilage of the rats with DDH. Therefore, DUSP26 may serve a potential synergistic protective role via the β-catenin pathway.

The core focus of the present study was on secondary osteoarthritis associated with DDH; however, the pathological processes of both primary and secondary osteoarthritis are centered on cartilage degeneration, and they share key pathological pathways such as abnormal chondrocyte phenotype, ECM metabolic disorder and activation of inflammatory pathways. The regulatory target (DUSP26) identified in the present study exerts its protective effect on secondary osteoarthritis by regulating the shared pathways. This mechanism suggests that its potential application can be extended to primary osteoarthritis, providing a theoretical basis for the broad-spectrum treatment of osteoarthritis. As a serine/threonine phosphatase containing a conserved catalytic domain, DUSP26 may be targeted in the future to delay osteoarthritis progression through the development of small-molecule activators (to enhance its catalytic activity). Additionally, based on the identified mechanism by which DUSP26 inhibits HDAC1/2/8, the combined use of DUSP26 activators and HDAC inhibitors is expected to exert a synergistic protective effect on chondrocytes.

Adenoviral vectors are widely used in the field of gene delivery, but they may themselves trigger the innate immune response of cells. To rule out interference from the vector itself on the experimental results, a control group of non-functional empty adenoviral vectors (Ad-EP or Ad-shNC) was specifically set up. Compared with in cells without adenoviral treatment, the expression levels of key cartilage markers (for example, COL2A1 and COL1A1) and pro-inflammatory cytokines (for example, IL-6 and TNF-α) in cells infected with Ad-EP or Ad-shNC were not significantly altered. This indicates that the empty adenoviral vectors had no impact on the relevant functional phenotypes of the cells. For shRNA-based gene silencing, the risk of off-target silencing caused by the complementarity between some sequences and non-target mRNAs was also taken into consideration. To eliminate this interference, two independent shRNAs (shDUSP26-1 and shDUSP26-2) were designed, which target non-overlapping regions of DUSP26 mRNA. Notably, both shRNAs effectively downregulated the protein and mRNA expression levels of DUSP26. A non-targeting shRNA (shNC) was used as a negative control, and it had no effect on DUSP26 expression or chondrocyte function, this further confirmed that these phenotypes were the results of shRNA specifically silencing the target gene. The aforementioned results confirm that the cell phenotypic changes mediated by DUSP26 in the present study were not caused by the off-target effects of the gene manipulation tools, but rather the specific outcome of changes in DUSP26 gene expression.

A limitation of the present study should be acknowledged. The results of the present study demonstrated that overexpressed DUSP26 induced a specific downstream phenotypic response (cartilage degradation) and, critically, drove dephosphorylation of its known substrates, HDAC1, HDAC2 and HDAC8. While the catalytic activity of DUSP26 upon overexpression is well-documented via the standard 'wild-type (with intact phosphatase activity) vs. phosphatase-dead mutant (with site-directed mutations in its catalytic domain that eliminate phosphatase activity but preserve protein structure)' control paradigm (14,32), and the present findings align with this framework, a direct in vitro phosphatase activity assay would have provided more definitive mechanistic evidence for the catalytic function of DUSP26. Future studies incorporating such direct measurements will strengthen the causal link between DUSP26 and its pro-degradative role in cartilage. Another limitation is the lack of clinical samples to evaluate the potential of DUSP26 as a biomarker for early diagnosis of DDH-associated secondary osteoarthritis. In DDH model rats, DUSP26 showed a compensatory increase when acetabular cartilage degenerated, this change preceded potential clinical symptoms (for example, joint pain and limited mobility). Compared with traditional imaging indicators, DUSP26 expression changes reflect chondrocyte stress earlier, offering a basis for 'subclinical diagnosis'. Thus, confirmation is needed in a human DDH cohort to determine whether DUSP26 expression is higher in patients with early DDH-associated secondary osteoarthritis than in healthy individuals.

In summary, the findings of the present study indicated that DUSP26 may inhibit cartilage damage via HDAC1/2/8 inactivation, supporting the future development of DUSP26-activating targeted agents (for example, DUSP26 agonists and HDAC1/2/8 inhibitor combinations) to intervene in early cartilage degeneration in patients with DDH, thus reducing secondary osteoarthritis incidence and improving long-term joint function. While further research is needed, the results of the present study provide valuable references for preclinical studies on DDH-associated secondary osteoarthritis.

Supplementary Data

Availability of data and materials

The mRNA-seq data generated in the present study may be found in the BioProject repository under accession number PRJCA050984 or at the following URL: https://ngdc.cncb.ac.cn/search/specific?db=bioproject&q=PRJCA050984. The other data generated in the present study may be requested from the corresponding author.

Authors' contributions

EW conceptualized the study, conducted investigation and wrote the original draft of the manuscript. HZ conceptualized the study, conducted investigation and formal analysis. DW conducted investigation. SA and XJ wrote, reviewed and edited the manuscript. XJ provided resources and acquired funding. SA and XJ performed the bioinformatics analysis and confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All animal experiments were carried out in accordance with Guide for the Care and Use of Laboratory Animals Eighth Edition 2011 (33) and were approved by the Animal Experiment Committee of Shengjing Hospital of China Medical University (approval no. 2025PS886K; Shenyang, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Natural Science Foundation of Liaoning (grant no. 2022-MS-188).

References