Adamts4 coordinates the transcriptomic profile of primary rat costal chondrocytes
Zhenxing Wei, Yijie Chen, Wanyi Kou, Yifan Zhang, Wenqi Sha, Ruixin Guo, Yuran Lei, Ningrui Zhang, Yanxia Shi, Zhenghui Wang
TL;DR
This study shows that Adamts4 influences gene expression in rat chondrocytes, possibly through interactions with RNA-binding proteins and specific mRNAs.
Contribution
The study reveals a novel role of Adamts4 in modulating chondrocyte transcriptomes via RNA-binding protein interactions and mRNA binding.
Findings
Adamts4 silencing altered genes related to cell differentiation and cycle progression.
Adamts4 interacts with RNA-binding proteins in rat chondrocytes.
Three genes (Hmox1, Acan, Col2a1) were deregulated and enriched in Adamts4 RIP samples.
Abstract
Chondrocytes play a pivotal role in cartilage tissue engineering. ADAMTS4 gene encodes aggrecanase-1, which is known to affect chondrocyte biology by regulating aggrecan degradation. However, the molecular mechanism by which ADAMTS4 regulates chondrocyte phenotype remains unclear. To comprehensively investigate Adamts4-regulated genes in primary rat costal chondrocytes, we conducted siRNA-mediated Adamts4 knockdown alongside RNA sequencing (RNA-seq), co-immunoprecipitation coupled with mass spectrometry (CO-IP/MS), and enhanced RNA immunoprecipitation sequencing (iRIP-seq). Our results demonstrated that Adamts4 knockdown did not affect chondrocyte apoptosis. However, Adamts4 silencing markedly changed the expression levels of numerous genes linked to cell differentiation and cell cycle progression. CO-IP/MS experiments showed that Adamts4 extensively interacted with RNA-binding proteins…
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Figure 5- —National Natural Science Foundation of China10.13039/501100001809
- —Funds for Shaanxi Province Zhong Dian Yan Fa Project
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Taxonomy
TopicsOsteoarthritis Treatment and Mechanisms · Mesenchymal stem cell research · Connective Tissue Growth Factor Research
Introduction
Cartilage injury is widespread in clinical practice. The regenerative and reparative abilities of articular tissue are limited, posing a challenge for restoring chondral and osteochondral tissues after injury in medical clinical research.1 The development of cartilage tissue engineering holds promise for repairing cartilage tissue defects.1^,^2 However, as potential seed cells of cartilage tissue engineering, chondrocytes have a de-differentiation phenotype during expansion.3^,^4 Some studies have explored how to regulate the de-differentiation process of chondrocytes.5^,^6 For example, increasing SOX9 expression can inhibit chondrocyte dedifferentiation, helping to maintain articular cartilage health.7 Understanding the key mechanisms for chondrocyte de-differentiation is vital for improving cartilage tissue engineering.
Cartilage tissue consists of a sparse population of chondrocytes embedded within an extracellular matrix (ECM), primarily composed of aggrecan and collagen, which is sustained by this limited cell number.8 An imbalance between the synthesis and breakdown of aggrecan disrupts normal cartilage function and can precipitate associated pathologies.9 Aggrecan is required for chondrocyte differentiation, and its reduction occurs during chondrocyte de-differentiation.5^,^6^,^10 The degradation of aggrecan mediated by aggrecanases represents an initial step in cartilage destruction observed in both rheumatoid arthritis and osteoarthritis.11–13 In particular, our previous studies reported that expression of genes encoding aggrecanases gradually increases with chondrocyte dedifferentiation,14 and their downregulation promotes chondrocyte-engineered cartilage formation by increasing aggrecan and total collagen.15
Two key aggrecanases, ADAMTS4 and ADAMTS5, are critically involved in aggrecan degradation16 and are considered potential therapeutic targets for osteoarthritis.17^,^18 Notably, undetectable ADAMTS4 (aggrecanase-1) activity in synovial fluid serves as a predictive biomarker for successful autologous chondrocyte implantation.19 Multiple studies have focused on the factors influencing the expression or aggrecanase activity of ADAMTS4. For instance, both fibronectin and CCN1 proteins can bind to the ADAMTS4 cysteine-rich domain and inhibit its aggrecanase activity in human cartilage.20^,^21 ADAMTS4 expression in human osteoarthritic chondrocytes were regulated by MicroRNA-125b.22 AQP1 depletion decreases ADAMTS4 expression in human osteoarthritis chondrocytes.23 However, few studies have systematically explored the ADAMTS4 downstream target genes in chondrocytes.
To gain deeper insight into the molecular functions of ADAMTS4 in chondrocyte biology, we employed Adamts4 knockdown, RNA sequencing (RNA-seq), co-immunoprecipitation with mass spectrometry (CO-IP/MS), and improved RNA-immunoprecipitation sequencing (iRIP-seq) in primary rat costal chondrocytes. Our results showed that Adamts4 knockdown modulated the transcriptomic profile of rat chondrocytes. Hmox1, Acan, and Col2a1 are three potential target genes for Adamts4. The interaction essential between RNA-binding proteins and target mRNAs may be an important mechanism underlying Adamts4’s gene regulatory activity in rat chondrocytes. These findings warrant further validation in animal models and human chondrocytes.
Methods
Isolation and culture of rat costochondral chondrocytes
Male Sprague-Dawley (SD) rats aged 4 weeks were supplied by the Animal Center of Xi’an Jiao Tong University (Xi’an, China). For the collection of the ribs medial to the costochondral junction, the rats were euthanized through CO_2_ inhalation. The rib cartilages were minced into small pieces and subjected to successive enzymatic digestion with 0.05% hyaluronidase for 0.5 h, 0.25% trypsin for 0.5 h, and 0.2% collagenase II (Sigma-Aldrich, St Louis, MO, USA) overnight at 37 °C. Following the removal of any undigested tissue, chondrocytes isolated from 10 rats were combined and grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). To avoid de-differentiation, the chondrocytes at passage 1 were used for this study.
Cell transfection and adenoviral transduction
For Adamts4 knockdown, three distinct siRNA duplexes and a nontargeting control siRNA were obtained from Gemma (Suzhou, China). Their sequences are provided in Supplementary Table S1 (see online supplementary material). Transfection of chondrocytes with Adamts4-targeting siRNAs was executed using Lipofectamine 2000 (Thermo Fisher Scientific) following the supplier’s instructions. Cells were collected 48 h post-transfection to evaluate Adamts4 expression levels.
Recombinant lentivirus carrying specific Adamts4 siRNAs was produced by GenePharma (Shanghai, China). Rat chondrocytes were subjected to lentiviral infection for 72 h, followed by selection with 5 μg/mL puromycin. Established stable cell clones were propagated in medium containing 2 μg/mL puromycin.
Assessment of Adamts4 knockdown
The Gapdh gene served as an internal control to evaluate knockdown efficiency. Standard protocols were used for cDNA synthesis. Real-time quantitative PCR (RT-qPCR) was conducted on a Bio-Rad CFX96 system using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd, China). Primer sequences are listed in Supplementary Table S1 (see online supplementary material). Transcript levels were normalized to Gapdh mRNA employing the 2^-ΔΔCT^ method.
Apoptosis analysis
To assess apoptosis, harvested cells were stained using an Annexin V-PE/7-AAD Apoptosis Detection kit. Apoptotic cells were quantified with a Beckman MoFlo XDP flow cytometer, and data analysis was performed using Flowjo (TreesStar) software.
Co-immunoprecipitation and mass spectrometry analysis
For immunoprecipitation, treated cells were lysed in TNE buffer (10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 2 mmol/L EDTA, 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Thermo Fisher Scientific). Lysates were immunoprecipitated with either an anti-ADAMTS4 antibody (catalog #: H00009507-D01; Abnova, Beijing, China) or a control IgG antibody (catalog #: 2729 s; Cell Signaling Technology) and subsequently incubated with protein A/G beads at 4 °C for 4 h. The immunoprecipitated complexes were washed, eluted by boiling, and visualized via silver staining using a Fast Silver Stain Kit (P0017S, Beyotime) as per the manufacturer’s guidelines. Subsequent mass spectrometry analysis was carried out by Novogene.
RNA extraction and sequencing
Total RNA was isolated using TRIZOL reagent (Thermo Fisher Scientific). The RNA underwent further purification through two rounds of phenol-chloroform extraction and was then treated with RQ1 DNase (Promega, Madison, WI, USA) to remove genomic DNA. RNA purity and concentration were reassessed by determining the A260/A280 ratio with a SmartSpec Plus spectrophotometer (Bio-Rad, USA). RNA integrity was confirmed by electrophoresis on a 1.5% agarose gel.
For each sample, 1 μg of total RNA was input for library construction with the VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme). Poly(A)+ mRNA was selected, fragmented, and converted to double-stranded cDNA. Following end repair and A-tailing, VAHTS RNA Adapters (Vazyme) were ligated. Ligated products within the 200-500 bp size range were purified, treated with heat-labile UDG, and the resulting single-strand cDNA was amplified, purified, quantified, and stored at −80°C until sequencing. Libraries were prepared according to the manufacturer’s protocol and sequenced on an Illumina HiSeq X Ten platform for 150 nt paired-end reads.
RNA-seq raw data processing and alignment
Sequencing reads containing over two ambiguous (N) bases were initially filtered out. Adaptor sequences and low-quality bases were then trimmed from the raw reads using the FASTX Toolkit (Version 0.0.13). Reads shorter than 16 nt after trimming were also discarded. The resulting high-quality reads were aligned to the mRatBN7.2 reference genome using TopHat2,24 allowing for four mismatches. Uniquely mapped reads were utilized to compute gene read counts and FPKM (fragments per kilobase of transcript per million mapped fragments) values.25
Differentially expressed genes analysis
Differentially expressed genes (DEGs) were identified using the edgeR package in the R Bioconductor environment.26 A false discovery rate (FDR) < 0.05 and an absolute fold change >2 were set as the threshold for defining significant differential expression.
iRIP-seq library preparation and sequencing
Rat chondrocytes were crosslinked once at 400 mJ/cm^2^ and lysed in ice-cold wash buffer. Lysis was performed in cold Wash buffer (1× PBS, 0.1% SDS, 0.5% NP-40, 0.5% sodium deoxycholate) supplemented with 200 U/mL RNase inhibitor (Takara) and a protease inhibitor cocktail (Roche) on ice for 30 min. RQ I DNase (Promega; 1 U/μL) was added and incubated at 37 °C for 30 min. A stop solution was introduced to halt DNase activity. The lysate was vigorously vortexed and centrifuged at 13 000 × g for 20 min at 4 °C to pellet cellular debris. RNA was then partially digested using MNase (Thermo Fisher Scientific).
For immunoprecipitation, the supernatant was incubated overnight at 4 °C with 10 μg of anti-ADAMTS4 antibody or control IgG. Immune complexes were subsequently captured by incubation with Protein A/G Dynabeads (Thermo Fisher Scientific) for 2 h at 4 °C. After magnetic separation and supernatant removal, the beads were washed twice sequentially with lysis buffer, high-salt buffer (250 mM Tris 7.4, 750 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% NP-40, 0.5% deoxycholate), and PNK buffer (50 mM Tris, 20 mM EGTA, 0.5% NP-40). Proteinase K (Roche) was used to digest proteins in both the 1% input sample and the immunoprecipitated samples. RNA was purified with Trizol reagent, and cDNA libraries were constructed using the KAPA RNA Hyper Prep Kit (KAPA, KK8541) following the manufacturer’s protocol. Final libraries were sequenced on an Illumina NovaSeq system.
Analysis of iRIP-seq
After aligning reads to the genome with TopHat2,24 only uniquely mapped reads were retained for subsequent analysis. The “ABLIRC” pipeline27 was applied to identify ADAMTS4 binding regions on the genome. Reads with any base pair overlap were merged into peaks. For each gene, a computational simulation generated random reads matching the number and length distribution of the observed peak reads. These simulated reads were then mapped back to the same genes to establish a random maximum peak height from overlapping reads. This process was iterated 500 times. Observed peaks with heights exceeding the random maximum peak height (P-value <0.05) were considered significant. Peaks identified in the IP sample that overlapped with peaks from the input control sample were excluded. Final target genes for ADAMTS4 were defined by the remaining significant peaks, and binding motifs were identified using HOMER software.28
Functional enrichment analysis
To determine the biological functions of DEGs, enrichment analyses for Gene Ontology (GO) terms and KEGG pathways were conducted using the KOBAS 2.0 server.29 The hypergeometric test was employed to assess enrichment significance, with multiple testing correction via the Benjamini–Hochberg FDR procedure.
Statistical analysis
Statistical analyses were performed using R software (v3.1.3). Comparisons between two groups were made using an unpaired two-tailed t-test. P values less than 0.05 were considered statistically significant.
Results
Silencing Adamts4 did not affect apoptotic level in rat primary costal chondrocytes
To study Adamts4’s effects on chondrocytes, primary rat costal chondrocytes were obtained and cultured in vitro. Collagen II is an early and abundant marker of chondrocyte differentiation.4 To detect the chondrocyte properties of costal chondrocytes, collagen II was detected by immunofluorescence in the cultured rat chondrocytes. It was shown that collagen II was highly expressed, especially in cytoplasm (Figure 1A), indicating that the rat costal chondrocytes retain chondrocyte properties.
*Silencing Adamts4 did not affect apoptotic level in primary rat costal chondrocytes. (A) Expression of Collagen II in third-generation costal chondrocytes. Red fluorescence indicates Collagen II, blue fluorescence indicates DAPI (nuclear). (B) Expression of Adamts4 in costal chondrocytes after transiently knocking down with three different small interfering RNA (siRNA). Error bars denote mean ± SEM (n = 3). (C) Adamts4 expression in chondrocytes after stable knockdown via lentiviral transduction. Error bars denote mean ± SEM. ***P-value < 0.0001. (D) Apoptosis analysis in Adamts4 knockdown cells compared to control cells (n = 3).
Then, Adamts4 was knocked down in the costal chondrocytes to study its biological function. Three different siRNAs (si278, si590, and si1298) were synthesized and transiently transfected into costal chondrocytes. Among them, the transient interference effect of siRNA 278 was the best (Figure 1B). Next, siRNA 278 was selected for lentivirus packaging. RT-qPCR detection showed Adamts4 was successfully knocked down by lentiviral transduction (Figure 1C).
Given that apoptosis is considered a key process in cartilage degradation,30 we assessed whether Adamts4 silencing influenced chondrocyte apoptosis. The results demonstrated that Adamts4 knockdown had a poor effect on chondrocyte apoptosis (Figure 1D), indicating that Adamts4 may not function through apoptosis.
Silencing Adamts4 globally changes the gene expression profile in rat costal chondrocytes
To identify the potential target gene of Adamts4 in rat costal chondrocytes, we performed RNA-seq on chondrocytes transfected with siAdamts4 or control vectors. Six RNA-seq libraries (three biological replicates per condition: siAdamts4_1-3, siCtrl_1-3) were constructed and sequenced. Gene expression levels, quantified as fragments per kilobase per million mapped reads (FPKM), were calculated for each sample. Principal component analysis (PCA) based on the FPKM values of all expressed genes revealed a clear distinction between the siAdamts4 and control groups (Figure 2A), demonstrating that Adamts4 silencing reshapes the gene expression profile in rat primary costal chondrocytes.
*Silencing Adamts4 regulates gene transcriptome profile in rat primary costal chondrocytes. (A) PCA plot based on FPKM values of all detected genes. Confidence ellipses are drawn for each group. (B) Volcano plot displaying all the DEGs between knockdown and control samples identified by edgeR (FDR < 0.05, |FC| ≥ 2). (C) Heatmap of hierarchical clustering showing expression levels of all the DEGs. (D and E) Bubble charts illustrating the most enriched GO biological processes for upregulated (D) and downregulated (E) DEGs. (F) Bar graph showing expression levels and statistical significance for selected DEGs. Error bars denote mean ± SEM (n = 3). **P-value < 0.001.
Differentially expressed genes (DEGs) between the two groups were identified using edgeR as outlined in the Methods. We found 397 upregulated and 314 downregulated genes in siAdamts4 cells compared to controls, indicating widespread transcriptional changes (Figure 2B). Hierarchical clustering of the normalized FPKM values for these DEGs showed clear separation between the two conditions and high reproducibility among replicates (Figure 2C), confirming that Adamts4 silencing significantly alters the expression of numerous genes.
Next, GO enrichment analysis was performed to elucidate the potential functions of the DEGs. Upregulated DEGs were significantly associated with biological processes such as transcriptional regulation, anatomical structure morphogenesis, potassium ion transmembrane transport, and cell differentiation (Figure 2D). Downregulated DEGs were enriched in processes including chromosome segregation, microtubule-based movement, mitotic cell cycle, cell division, mitotic nuclear division, and regulation of cell growth (Figure 2E). For instance, there were higher expression level for Acan gene encoding aggrecan, and several genes (Hmox1, Gdf15, Nr4a1, and Dusp5) associated with cartilage degeneration in siAdamts4-transfected cells than that of control cell (Figure 2F). On the contrary, the Col2a1 gene encoding collagen, Fgf18, Igfbp4, Itgbl1, and Nog had a lower expression level after Adamts4 knockdown (Figure 2F). Taken together, these results demonstrated that Adamts4 depletion regulates multiple genes associated with cell cycle in rat costal chondrocytes.
Adamts4 interacts with RBPs in rat costal chondrocytes
To identify Adamts4-interacting proteins, a Co-IP assay was performed in rat costal chondrocytes using normal IgG and anti-Adamts4 antibody. Silver staining showed that the Adamts4 group had more protein bands compared to the IgG group, indicating the Adamts4-interacting proteins (Figure 3A). Mass spectrometry identified 298 proteins in the Adamts4 group and 147 in the IgG group, with 199 proteins uniquely detected in the Adamts4 pull-down (Figure 3B).
Identification of ADAMTS4-associated proteins by co-immunoprecipitation (Co-IP)-based proteomics. (A) PAGE gel silver staining image of the Adamts4 and IgG groups. (B) Venn diagram showing the overlap of proteins identified in the ADAMTS4 and IgG groups. (C) GO analysis of Adamts4-associated proteins. (C) KEGG analysis of Adamts4-associated proteins. (E) Classification of Adamts4-associated proteins.
KEGG and GO enrichment analyses were conducted to understand the functions of these Adamts4-associated proteins. GO analysis indicated enrichment in terms such as translation, mRNA splicing via spliceosome, mRNA processing, and osteoblast differentiation (Figure 3C). KEGG analysis highlighted pathways including ribosome, spliceosome, and focal adhesion (Figure 3D). Considering that multiple enriched pathways are those related to RNA processing, we further overlapped the Adamts4-associated proteins with a curated list of RBPs and transcription factors. The results showed that 107 of 199 (53.77%) Adamts4-associated proteins were RBPs (Figure 3E).
ADAMTS4 interacts with and regulates Hmox1, acan, and Col2a1 in rat costal chondrocytes
To further explore whether Adamts4 affects gene expression by directly interacting with target RNAs, we employed iRIP with Adamts4 in rat costal chondrocytes. Western blotting confirmed the presence of ADAMTS4 protein specifically in the anti-ADAMTS4 immunoprecipitate (Figure 4A). Sequencing libraries were prepared from the immunoprecipitated RNA and an input control. Mapping of the clean reads showed that Adamts4-bound reads were predominantly enriched in 3ʹ UTRs, coding sequences (CDS), and intronic regions (Figure 4B). Using the ABLIRC pipeline,27 we identified 4523 peaks (corresponding to 1834 genes) and 3503 peaks (1331 genes) in two independent replicates, respectively, after filtering out peaks common to the input control. Motif analysis with HOMER revealed that an AU-rich motif was the most significantly enriched within ADAMTS4-bound peaks in both replicates (Figure 4C and D). Example read density profiles displaying ADAMTS4 binding on the Eif3a, Ncl, and Ccnd1 genes are shown (Figure 4E-G).
ADAMTS4 binds to mRNAs of genes associated with chondrocyte de-differentiation in rat chondrocytes. (A) Western blot verifying the efficiency of immunoprecipitation. (B) Pie chart depicting the genomic distribution of ADAMTS4 binding peaks. (C) Top five sequence motifs enriched in ADAMTS4-bound peaks, identified by HOMER. (D) Bar graph showing the proportion of peaks containing an AU-rich motif. (E-G) IGV-sashimi plots illustrating ADAMTS4 binding peaks and read densities across the Eif3a (E), Ncl (F), and Ccnd1 (G) genes. Gene models are shown below the plots.
Then, we explored whether Adamts4 regulates expression at the transcript level by binding to target genes. Overlap analysis showed that 45 and 37 Adamts4 silencing*-*induced DEGs were Adamts4-bound genes in two replicates, respectively (Figure 5A and B). In particular, the reads density landscape showed that AAdamts4 had at least one binding site on the mRNAs Hmox1, Acan, and Col2a1. Moreover, the expression level of the three genes increased or decreased after Adamts4 knockdown. Therefore, these results suggest that Adamts4 may regulate target genes by directly interacting with their transcripts.
*ADAMTS4 binds to mRNAs and regulates their expression in rat chondrocytes. (A and B) Venn diagrams showing the overlap between genes bound by ADAMTS4 (Peak genes) and differentially expressed genes (DEGs) in two independent replicates. (C-E) IGV-sashimi plots display Adamts4 binding peaks and read densities across the Hmox1 (C), Acan (D), and Col2a1 (E) genes. Corresponding bar graphs (right panels) show the expression changes of these genes upon Adamts4 knockdown. Error bars denote mean ± SEM (n = 3). **P-value < 0.001.
Discussion
Chondrocytes are an essential player in cartilage tissue engineering.3^,^4 Exploring the underlying chondrocyte de-differentiation will provide a basis for the development of new cartilage engineering materials. The ADAMTS4 gene shows a gradual increase in expression with chondrocyte dedifferentiation. Knockdown of ADAMTS4 promotes chondrocyte-engineered cartilage formation.14^,^15 In addition, some proteins could interact with ADAMTS4 and inhibit its aggrecanase activity in human cartilage.20^,^21 Thus, ADAMTS4 plays a significant role in modulating the chondrocyte phenotype. However, the interacting proteins and downstream regulatory target genes of ADAMTS4 remained unclear. Our study provides evidence that Adamts4 can influence gene expression in rat chondrocytes, potentially through direct RNA binding or association with RBPs. The results provide new clues for the further study of the function and mechanism of ADAMTS4 in human chondrocyte phenotype.
Previous studies show that knockdown of Adamts4 or inhibition of ADAMTS4 protein activity promotes chondrocyte proliferation.15^,^20 In smooth muscle cells, ADAMTS4 can enter the nucleus under stress and cleave PARP-1, leading to apoptosis.31 In addition, overexpression of ADAMTS4 significantly promotes fibroblast apoptosis.32 In this study, knockdown of the Adamts4 gene did not considerably affect chondrocyte apoptosis. We speculated that the effect of ADAMTS4 on apoptosis may be cell-type specific.
As far as we know, many studies focus on the factors that regulate the expression of the ADAMTS4 gene. For instance, several microRNAs target the ADAMTS4 gene and regulate its expression.22^,^32–34 The application of specific biochemical agents either upregulated or downregulated the expression levels of ADAMTS4 at both the mRNA and protein levels.35–37 However, systematic identification of its downstream transcriptional targets has been lacking. We present the first genome-wide identification of potential downstream targets regulated by the ADAMTS4 gene. Transcriptomic analysis showed that silencing Adamts4 significantly altered the expression of 711 genes. In particular, functional enrichment analysis indicated that ADAMTS4-regualted genes included Hmox1, Dusp5, Nr4a1, Fgf18, and Nog. These genes play essential roles in regulating osteogenic differentiation in different cells under various conditions.38–42 For instance, FGF18 inhibits osteogenic differentiation in mouse osteoblast precursors via the ERK pathway.41 Thus, future studies should examine whether ADAMTS4 influences chondrocyte de-differentiation by modulating these genes.
It is well-established that interacting proteins like fibronectin and CCN1 can inhibit ADAMTS4’s aggrecanase activity.20^,^21 Generally, a protein needs to interact with other proteins to exert its biological functions in physiology and pathology, and the protein–protein interactions (PPIs) are potential therapeutic targets.43–45 Here, we systematically identified, for the first time, the potential interacting proteins of ADAMTS4. In particular, our study showed that ADAMTS4 could bind 107 RBPs. Some proteins lack canonical RNA-binding domains, showing accidental RNA interactions that are rarely sequence-specific.46–48 Based on the interaction of ADAMTS4 and RBPs, iRIP-seq was performed to identify RNAs bound by ADAMTS4. The results showed that ADAMTS4 could bind RNAs with a biased consensus AU-rich motif. In fact, KH-type splicing regulatory protein (KSRP), ELAV/Hu family proteins, and tristetraprolin (TTP) family proteins show RNA-binding potential due to AU-rich motifs.49–51 These data imply that ADAMTS4 may function as an atypical RBP, either binding target RNAs directly or cooperating with other RBPs to regulate gene expression.
RBPs are pivotal post-transcriptional regulators, controlling mRNA stability and translation.52 In this study, we found that many genes (e.g., Hmox1, Acan, and Col2a1) were significantly regulated upon Adamts4 knockdown in rat chondrocytes. The HMOX1 gene encoding heme oxygenase-1 plays essential roles in cartilage degeneration and ECM degradation in chondrocytes.53^,^54 The encoded proteins of the Acan and Col2a1 genes are aggrecan and collagen, respectively, which are primary components of the ECM in cartilage tissue. Downregulation of aggrecan expression facilitates dedifferentiation of chondrocytes and cartilage destruction.55 SOX9 regulates COL2A1 expression during chondrocyte differentiation,56 and the transcription factor C-Krox differentially controls the COL2A1 enhancer in differentiated versus dedifferentiated states.57 These observations suggest that Hmox1, Acan, and Col2a1 may mediate ADAMTS4’s effects in chondrocytes.
However, several limitations of our study should be acknowledged. First, the present findings are based on rat chondrocytes. Further studies should be validated in animal models and human chondrocytes. Second, direct functional evidence linking Adamts4 to the regulation of chondrocyte de-differentiation is still needed. Finally, ADAMTS4 can modulate a lot of genes other than Hmox1, Acan, and Col2a1 in chondrocytes. The detailed mechanism for Adamts4-dependent regulation of gene expression warrants further investigation.
In summary, our data unveil the transcriptomic changes elicited by Adamts4 silencing in rat chondrocytes. Adamts4 may act as a novel RBP, binding target mRNAs or interacting with other RBPs to modulate downstream gene expression. Future research should focus on identifying the key downstream genes through which Adamts4 governs chondrocyte dedifferentiation and evaluating its therapeutic potential in cartilage tissue engineering.
Supplementary Material
szag004_Supplementary_Data
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