Deficiency of Krüppel-like factor 15 activates β-catenin and is involved in the development of osteoarthritis

doi:10.21203/rs.3.rs-8429330/v1

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Deficiency of Krüppel-like factor 15 activates β-catenin and is involved in the development of osteoarthritis

https://doi.org/10.21203/rs.3.rs-8429330/v1

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Krüppel-like factor 15 is a metabolism-related transcription factor that has been implicated in the regulation of the Wnt/β-catenin signaling pathway. This study aimed to elucidate its function in cartilage, particularly its interaction with β-catenin, using inducible cartilage-specific knockout mice. Krüppel-like factor 15 knockout mice and control mice underwent destabilization of medial meniscal surgery to induce osteoarthritis. Histological analysis and immunohistochemistry were carried out, along with in vitro co-immunoprecipitation, double immunofluorescence, and subcellular fractionation. The effects of Wnt3A stimulation and treatment with Wnt/β-catenin inhibitors were evaluated. KO mice showed significantly more cartilage degeneration at eight weeks after surgery when compared with control mice. Knockout mice also showed elevated Osteoarthritis Research Society International scores and increased expression of β-catenin, matrix metalloprotease 13, and a disintegrin-like metalloprotease with thrombospondin motifs 5, along with decreased expression of SRY-box transcription factor 9. Krüppel-like factor 15 was seen to physically interact and colocalize with β-catenin in chondrocyte nuclei. Its deficiency promoted Wnt/β-catenin pathway activation and nuclear β-catenin accumulation. Wnt3A stimulation amplified catabolic gene expression and suppressed anabolic factor expression. However, administration of a Wnt/β-catenin signaling inhibitor canceled out these effects. This study indicates that Krüppel-like factor 15 interacts with β-catenin and functions as a negative regulator of Wnt/β-catenin signaling in cartilage, maintaining matrix homeostasis, and suppressing osteoarthritis progression. Deficiency of Krüppel-like factor 15 promotes catabolism and inhibits anabolism mediated by β-catenin in cartilage. These findings suggest that Krüppel-like factor 15 may be a potential therapeutic target for osteoarthritis, either by restoring its function or enhancing its interaction with β-catenin.

Krüppel-like factor 15

Wnt/β-catenin pathway

β-catenin

osteoarthritis

destabilization of medial meniscal surgery

Wnt3A

matrix metalloprotease 13

disintegrin-like metalloprotease with thrombospondin motifs 5

SRY-box transcription factor 9

Osteoarthritis (OA) is a common joint disease that develops due to aging and lifestyle habits, which significantly reduces quality of life.^1,2 The pathology of OA progresses as a series of complex changes in the entire joint structure, accompanied by degeneration of articular cartilage, synovitis, bone sclerosis, and osteophyte formation. To date, no effective disease-modifying treatment has been established to treat OA.^3.4 Artificial joint replacement surgery has delivered good long-term results.⁵ However, the efficacy of conservative treatments such as analgesics, hyaluronic acid injections, and rehabilitation is limited in their efficacy.⁶ Therefore, elucidating the mechanisms underlying OA progression and identifying new therapeutic targets is necessary.

Krüppel-like factors (KLFs) are a family of transcription factors with C2H2-type zinc fingers that are involved in a wide range of biological processes.⁷ Currently, 17 members of the KLF family have been identified, each with different tissue specificities and functions.^8,9 Recently, the involvement of KLFs in OA has attracted attention. In particular, KLF2 and KLF4, which are involved in the maintenance of chondrocyte homeostasis, are known to be associated with OA pathology.¹⁰ KLF2 inhibits oxidative stress and ferroptosis and protects mitochondrial function in chondrocytes, thereby suppressing OA progression. Specifically, KLF2 inhibits the oxidative stress response by activating Nrf2/ARE signaling.¹¹ It improves ferroptosis and mitochondrial dysfunction via the SIRT1/GPX4 pathway, thereby alleviating OA pathology.¹² KLF4 promotes chondrocyte differentiation and expression of extracellular matrix (ECM) components, and is considered an important transcription factor in cartilage repair regulation. Furthermore, KLF4 has a protective function in OA by regulating the inflammatory response of synovial cells and chondrocytes and suppressing tissue destruction.^13,14

KLF15 is a metabolism-related transcription factor that functions as a key regulator of metabolic homeostasis in various tissues, specifically in lipid metabolism and energy homeostasis.^15–18 In skeletal muscle, KLF15 cooperates with peroxisome proliferator-activated receptor delta (PPARδ) to regulate lipid flux and maintain mitochondrial function, contributing to exercise response and muscle function adaptation.¹⁹ However, the possibility that KLF15 may also be involved in the pathology of OA has recently attracted attention. Disruption of lipid metabolism is considered a new pathological factor involved in the disturbance of cartilage homeostasis and OA progression.²⁰ Abnormal fatty acid oxidation impairs cartilage homeostasis through SRY-box transcription factor 9 (SOX9) degradation and epigenetic regulation.²¹ Such metabolic abnormalities promote cartilage degeneration. Therefore, KLF15 may function as a metabolic regulator even within chondrocytes. KLF15 directly induces SOX9 expression during chondrogenic differentiation of human mesenchymal stem cells (hMSCs) and promotes cartilage differentiation.²² Additionally, KLF15 expression is significantly reduced in chondrocytes derived from patients with OA compared with healthy individuals.¹⁰ Furthermore, KLF15 plays a role in suppressing the inflammatory response and progression of matrix degradation by suppressing matrix metalloproteinase-13 (MMP-13) transcription in response to tumor necrosis factor-alpha (TNFα) stimulation.²³ Our previous study reported that KLF15 deficiency reduces peroxisome proliferator-activated receptor gamma (PPARγ) expression, which promotes chondrocyte apoptosis, resulting in the worsening of OA pathology.²⁴ Thus, KLF15 is a multifaceted transcription factor that contributes to the metabolic regulation, differentiation control, and suppression of inflammatory responses in cartilage. Specifically, its role in homeostasis is extremely important. However, the role of KLF15 in cartilage homeostasis and the progression of OA pathology has not yet been fully elucidated, and further research is required.

The Wnt/β-catenin signaling pathway is a highly conserved and fundamental signaling cascade that plays pivotal roles in several biological processes.²⁵ Canonical Wnt signaling through β-catenin is activated by Wnt ligand binding to the cell surface Frizzled receptor and co-receptor low-density lipoprotein receptor-related protein (LRP) 5/6. In the absence of Wnt ligands, cytoplasmic β-catenin is targeted for proteasomal degradation via a degradation complex, which phosphorylates β-catenin and marks it for ubiquitination. Upon Wnt activation, this degradation machinery is inactivated and β-catenin accumulates in the cytoplasm before translocating to the nucleus. There, Wnt/β-catenin activates the transcription of various target genes involved in cell proliferation, differentiation, and matrix remodeling.²⁶ The Wnt/β-catenin pathway is essential for proper cartilage tissue development and maintenance under physiological conditions. Its dysregulation has been suggested to be deeply involved in OA pathology. Evidence suggests that abnormal or persistent activation of this pathway promotes the expression of degradative enzymes, such as MMP13 and disintegrin-like metalloprotease with thrombospondin motifs 5 (ADAMTS5), which are central mediators of ECM degradation in OA cartilage.^27–29 These enzymes degrade collagen type II alpha 1 (Col2A1) and aggrecan, resulting in the structural destruction of articular cartilage. In particular, overexpression of β-catenin has been reported to be involved in the progression of OA.^30,31 Interestingly, recent studies suggest that transcription factors, such as KLF15, which regulate metabolic and signaling pathways in other tissues, such as the heart and kidney, may interact with Wnt/β-catenin signaling.^17,32 These findings suggest that KLF15 may also affect cartilage integrity by regulating the expression of β-catenin in chondrocytes.

Information regarding the crosstalk between KLF15 and β-catenin in OA pathology is currently scarce. Understanding the precise nature of the involvement of KLF15 in the Wnt/β-catenin pathway may help to identify novel transcriptional regulatory mechanisms in OA and aid in identifying future therapeutic targets. In this study, we aimed to evaluate how the interaction between KLF15 and β-catenin regulates OA progression using a KLF15-deficient mouse model.

Generation of inducible cartilage-specific KLF15 knockout mice

Mice were maintained under a 12-h light/dark cycle with free access to food and water. Klf15^fl/fl mice were generated as described previously.³³ Col2-CreERT2 mice were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). Klf15^fl/fl mice were crossed with Col2-CreERT2 mice to generate inducible KLF15 knockout mice (Klf15^fl/fl Cre mice). Genotyping was performed on tail biopsy samples by polymerase chain reaction (PCR) using a DNA extraction kit (Qiagen, Valencia, California, USA). Klf15^fl/fl littermates negative for Col2-CreERT2 were used as wild-type controls. To induce gene deletion, 6-week-old Klf15^fl/fl Cre mice and control mice were intraperitoneally injected with 4-hydroxytamoxifen (40 mg/kg) for 5 consecutive days. In this study, only male mice were used because they develop more severe OA than females.³⁴ All mice were backcrossed to the C57BL/6 background for more than six generations. Each experimental group included at least six mice.

In vivo OA mouse model

All animal procedures were conducted in strict accordance with the National Institutes of Health guidelines for the care and use of laboratory animals (Bethesda, MD, USA). The Animal Research Committee of Kobe University approved the protocol (approval number: P230606). Ten-week-old male Klf15^fl/fl Cre and control mice were used. All surgical procedures were performed on the right knee joint under combined anesthesia (medetomidine, 0.375 mg/kg; midazolam, 2.0 mg/kg; butorphanol, 2.5 mg/kg). OA was induced by destabilization of the medial meniscus (DMM) via medial meniscotibial ligament.³⁵ Sham operations involved exposure without ligament transection. Mice were allowed to bear weight after recovery. Mice were euthanized for histological evaluation at 1 day, 1 week, 4 weeks, and 8 weeks post-operation or at 8 weeks post-sham. Six mice were analyzed per group (total n = 60; KLF15 KO Sham, KLF15 KO DMM, Control Sham, Control DMM).

Histological evaluation of cartilage degeneration

Knee joints were fixed in 10% neutral-buffered formalin for 24 h, decalcified with 4% ethylenediaminetetraacetic acid for 7 days, and embedded in paraffin. Coronal sections were cut at 80 µm intervals and stained with Safranin O and Fast Green. OA severity was evaluated using the Osteoarthritis Research Society International (OARSI) cartilage histopathology scoring system.

Immunohistochemistry

Deparaffinized sections were digested for 10 min with proteinase (Dako, Glostrup, Denmark) and treated with 3% hydrogen peroxide (Wako Pure Chemical Industries, Osaka, Japan) to block endogenous peroxidase activity. Sections were incubated overnight at 4°C with the following primary antibodies: anti-β-catenin (1:5000, bs-0530R, Abcam, Cambridge, UK), anti-ADAMTS5 (1:50, GTX100332, GeneTex, Irvine, CA, USA), anti-MMP13 (1:50, GTX100665, GeneTex), and anti-SOX9 (1:200, ab185230, Abcam, Cambridge, UK). Secondary antibody incubation was performed using peroxidase-labeled antirabbit immunoglobulin (Histofine Simple Stain MAX PO; Nichirei Biosciences, Tokyo, Japan) at 22 ± 2℃ for 30 min, followed by staining with 3,3'-diaminobenzidine (DAB; Histofine Simple Stain DAB kit; Nichirei Biosciences, Tokyo, Japan). One coronal section at the center of the most severe OA lesion of each tibial plateau was analyzed. The number of positively stained cells in both superficial and deep zones of cartilage was counted in three high-power fields by three blinded observers. The mean percentage of positive cells to total cells was calculated for each marker per group (n = 6). Cells above the tidemark were considered positive.

Isolation of mouse cartilage and chondrocytes

Cartilage tissues were isolated from fetal rib cages. Klf15^fl/fl Cre mice were mated, and pregnant female mice were intraperitoneally injected with tamoxifen at 12.5 and 13.5 days post coitum (dpc). Fetuses were harvested from these mice by Caesarean section at 18.5 dpc. Genotyping was performed on tail biopsy samples by PCR using a DNA extraction kit (Qiagen, Valencia, California, USA). Primary chondrocytes were isolated from cartilage by digestion with 3 mg/mL collagenase D (Roche, Basel, Switzerland) in Dulbecco’s Modified Eagle’s Medium (DMEM; D5546, Sigma-Aldrich, St. Louis, MO, USA) at 37℃ under 5% CO₂ for 45 min. The digestion was repeated after transferring the cartilage pieces to a new dish. The resulting cells were cultured overnight in a 1:5 mix of culture medium (DMEM supplemented with 2 mM glutamine, 50 U/mL penicillin, 0.05 mg/mL streptomycin) and digestion solution. Non-adherent cells were removed, and adherent cells were further cultured with 10% fetal bovine serum (FBS; 50 mL per 500 mL DMEM) for 5–6 days until confluence. First-passage cells were used in all experiments.

Co-immunoprecipitation

Primary cultured chondrocytes from control fetal costal cartilage were used. Cells were lysed in M-PER cell lysis buffer (#78501, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). β-catenin antibody (1:5000, bs-0530R, Abcam, Cambridge, UK) was added to the cell lysate and incubated for 10 min at room temperature with rotation, followed by the addition of Dynabeads Protein A (10001D, Thermo Fisher Scientific, Waltham, MA, USA) with rotation for 1 h at 4℃ to precipitate the antigen-antibody complexes. After washing the beads twice with washing buffer, the precipitated complex was eluted, separated by SDS-PAGE, and detected by western blot with KLF15 antibody (1:500, ab2647; Abcam, Cambridge, UK). For the negative control (IP: IgG), normal rabbit IgG (same species and isotype as the primary antibody) was added to an identical amount of lysate and processed in parallel with Dynabeads Protein A under the same conditions. Whole cell lysates were also analyzed as input to confirm protein expression.

Double immunofluorescence

To confirm the colocalization of KLF15 and β-catenin in chondrocytes, fluorescent immunostaining was performed in chondrocytes from control mice (n = 5). Sections were incubated with 3% hydrogen peroxide (Wako Pure Chemical Corporation, Osaka, Japan) for 30 min. The sections were incubated overnight at 4℃ with anti-KLF15 antibody (1:500, ab2647; Abcam, Cambridge, UK) and anti-β-catenin antibody (1:5000, bs-0530R; Abcam, Cambridge, UK) as primary antibodies, and then incubated with the corresponding secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse, 1:500; A-11001, Alexa Fluor 568-conjugated rabbit anti-mouse, 1:500; A-11061, Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at room temperature in the dark. For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated at room temperature for 5 min. The cells were imaged using a confocal microscope.

Cytoplasmic and nuclear protein extraction

To compare β-catenin expression levels in cytoplasmic and nuclear fractions, western blot analysis was performed following subcellular fractionation. Primary chondrocytes were isolated from fetal costal cartilage of control and KLF15 knockout (KLF15 KO) mice. Cytoplasmic and nuclear proteins were extracted using homemade buffers (Buffer A and Buffer C). Buffer A consisted of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Buffer C consisted of 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF. Protease and phosphatase inhibitors were added immediately before use. Briefly, chondrocytes were lysed in ice-cold Buffer A for 15 min. IGEPAL CA-630 (CAS No. 9002-93-1) was then added, and the lysates were centrifuged at 12,000 rpm for 30 s at 4°C. The resulting supernatants were collected as the cytoplasmic fractions. The remaining pellets were resuspended in ice-cold Buffer C, followed by centrifugation at 12,000 rpm for 5 min at 4°C to obtain the nuclear fractions.

Western blot analysis

Chondrocytes (n = 4) were lysed using the NucleoSpin RNA/Protein kit (Takara Bio, Tokyo, Japan), and the total protein concentration was quantified using the Bradford method and protein assay reagent (Bio-Rad). Anti-β-catenin antibody (1:5000, bs-0530R, Abcam, Cambridge, UK) was used as the primary antibody. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (IgG Ab, GE Healthcare, Little Chalfont, UK) was used as the secondary antibody, and signals were visualized using ECL Plus reagent (GE Healthcare) on a Chemilumino Analyzer LAS-3000 mini (FujiFilm, Tokyo, Japan). As internal controls, rabbit polyclonal anti-Histone H3 antibody (1:8000; 17168-1-AP, Proteintech) was used for the nuclear fraction, and mouse monoclonal anti-GAPDH antibody (1:50000; 60004-1-Ig, Proteintech, Chicago, IL, USA) was used for the cytoplasmic fraction, and these were used to correct the purity and expression levels of the fractions. Protein expression was determined by semi-quantification of digitally captured images using NIH ImageJ software (http://imagej.nih.gov/ij/).

Quantitative RT-PCR analysis

Chondrocytes were cultured in 6-well plates for 24 h with or without stimulation by recombinant mouse Wnt3A protein (200 ng/mL, ab81484, Abcam, Cambridge, UK) and/or XAV-939 (200 ng/mL, S1180, Selleck Chemicals, Houston, TX, USA). Total RNA was extracted using QIAshredder and RNeasy Mini Kit (Qiagen, Valencia, California, USA). In detail, 1 µg of total RNA was reverse transcribed into first-strand cDNA using 1.25 µM oligo dT primer in 40 µL PCR buffer II containing 2.5 mM MgCl2, 0.5 mM dNTP mix, 0.5 U RNase inhibitor, and 1.25 U MuLV reverse transcriptase (PerkinElmer, Foster City, CA, USA) for 60 min at 42℃. Relative expression of β-catenin, Axin-like protein or conductin (Axin2, ADAMTS5), MMP13, SOX9, and the gene Col2A1, which encodes the alpha chain of type II collagen, was quantified by SYBR Green real-time PCR using an ABI Prism 7500 system (Applied Biosystems, Foster City, CA, USA). The relative expression of the target genes was normalized to that of the GAPDH housekeeping gene using the comparative cycle threshold (Ct) value method. The difference in the mean Ct value of the target gene and the housekeeping gene is shown as ΔCt, and the difference between ΔCt and the Ct value of the calibration sample is shown as ΔΔCt. Log2(ΔΔCt) represented the relative value of gene expression. Primer sequences are listed in Supplementary Figure S1.

Statistical analyses

Two-way analysis of variance (ANOVA) was used for statistical analysis, followed by Tukey’s post hoc test for multiple comparisons. Mann–Whitney U tests were used for comparisons between two groups. Statistical analyses were performed using EZR (Easy R), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). Results were expressed as means with 95% confidence intervals (CI). A p-value < 0.05 was considered statistically significant.

KLF15 knockout mice are more susceptible to OA-related changes in vivo

At 8 weeks postoperatively, KLF15 KO mice exhibited greater cartilage erosion and loss of Safranin O staining compared with control mice (Fig. 1A). According to the OARSI cartilage OA histopathology scoring system, the mean total scores in both KLF15 KO and control mice were significantly higher at 4 and 8 weeks after DMM surgery compared with sham-operated mice (Fig. 1B; KLF15 KO mice: p = 0.0086 and p = 0.0021, respectively; control mice: p = 0.0411 and p = 0.0021, respectively). The mean total score was significantly higher in KLF15 KO mice than in control mice at both 4 and 8 weeks postoperatively (p = 0.0259 and p = 0.0259, respectively).

KLF15 KO mice are more susceptible to changes in OA due to increased expression of β-catenin

In KLF15 KO mice, β-catenin expression was increased compared to control mice (Fig. 2A). The positive cell ratio of β-catenin was significantly increased in KLF15 KO mice compared with control mice at 4 and 8 weeks after surgery (p = 0.0259 and p = 0.0259, respectively; Fig. 2B). It was also significantly increased in both KLF15 KO and control mice at 4 and 8 weeks after DMM surgery compared with sham-operated mice (Fig. 2B, p = 0.0086 and p = 0.0021, respectively, in KLF15 KO mice, and p = 0.0411 and p = 0.0021, respectively, in control mice).

KLF15 KO mice exhibit increased catabolic factor expression in cartilage

KLF15 KO mice showed higher MMP13 and ADAMTS5 expression compared with control mice (Fig. 3A, C). In KLF15 KO mice, the positive cell ratio of MMP13 was significantly increased at 4 and 8 weeks after surgery, and the positive cell ratio of ADAMTS5 was significantly increased at 1, 4, and 8 weeks after surgery (Fig. 3B, D; MMP13: p = 0.0303 and p = 0.0259, respectively; ADAMTS5: p = 0.0043, p = 0.0021, and p = 0.0021, respectively). Compared to sham mice, the positive cell ratio of MMP13 was significantly increased in KLF15 KO mice at 4 and 8 weeks after surgery and in control mice at 8 weeks after surgery (KLF15 KO: p = 0.0303 and p = 0.0021, respectively; control: p = 0.0043). The positive cell ratio of ADAMTS5 was significantly increased in KLF15 KO mice at 1, 4, and 8 weeks and was similarly increased in control mice at 8 weeks after DMM surgery compared with sham mice (KLF15 KO: p = 0.0269, p = 0.0001, and p < 0.0001, respectively; control: p = 0.0043).

KLF15 KO mice exhibit decreased SOX9 expression, contributing to OA progression

SOX9 expression was reduced in KLF15 KO mice compared with that in control mice (Fig. 3E). The positive cell ratio of SOX9 was significantly decreased in KLF15 KO mice compared with control mice at all observation time points (Fig. 3F; p = 0.0086, p = 0.0086, p = 0.0259, p = 0.0086, and p = 0.0151, respectively). The positive cell ratio of SOX9 was significantly decreased in both KLF15 KO and control mice compared to sham mice at 4 and 8 weeks after DMM surgery (KLF15 KO: p = 0.0014 and p < 0.0001, respectively; control: p = 0.0016 and p < 0.0001, respectively).

KLF15 interacts with β-catenin in the nucleus of chondrocytes, while KLF15 deficiency activates the Wnt/β-catenin pathway

Co-immunoprecipitation was performed in chondrocytes from control mice to examine whether KLF15 interacts directly with β-catenin. Chondrocyte cell lysates were immunoprecipitated with β-catenin, and KLF15 was detected by western blotting. A clear band of the expected size for KLF15 was detected, confirming the interaction between KLF15 and β-catenin (Fig. 4A). Immunofluorescence staining revealed that KLF15 and β-catenin stained more intensely at the same position in the nucleus, confirming that KLF15 and β-catenin colocalize in chondrocyte nuclei (Fig. 4B).

Real-time RT-PCR analysis showed that β-catenin and Axin2 mRNA expression in chondrocytes derived from KLF15 KO mice was significantly higher than that in control mice (Fig. 4C, p = 0.0003 and p = 0.0357, respectively). KLF15KO and control mouse chondrocytes were separated by cell fractionation, and the expression levels of β-catenin protein in the cytoplasm and nucleus were detected by western blotting. There was no significant difference in the expression level of β-catenin in the cytoplasm between KLF15KO and control mice (Fig. 4D, 4E: p = 0.7023). The expression level of β-catenin in the nucleus was significantly higher in KLF15KO mice than in control mice (Fig. 4F, 4G: p < 0.0001).

Real-time RT-PCR of the Wnt/β-catenin pathway, catabolic factors, and anabolic factors following Wnt3A stimulation and XAV939 treatment in KLF15 KO mouse chondrocytes

Real-time RT-PCR analysis showed that Wnt3A stimulation significantly increased the expression of β-catenin, Axin2, MMP13, and ADAMTS5 in both KLF15KO and control mice (β-catenin, p < 0.0001 and p < 0.0001, respectively; Axin2, p < 0.0001 and p < 0.0001, respectively; MMP13, p = 0.0027 and p = 0.0005, respectively; ADAMTS5, p < 0.0001 and p < 0.0001, respectively). Furthermore, the increase in expression of these four genes by Wnt3A stimulation was canceled by XAV939 treatment in both KLF15KO and control mice, and their expression was significantly decreased (β-catenin, p < 0.0001 and p < 0.0001, Axin2, p < 0.0001 and p < 0.0001; MMP13, p = 0.0045 and p = 0.0045, respectively; ADAMTS5, p < 0.0001 and p < 0.0001, respectively). Wnt3A stimulation significantly decreased the expression of SOX9 and Col2A1 in both KLF15KO and control mice (all p < 0.0001). Furthermore, the decrease in the expression of these two genes by Wnt3A stimulation was canceled by XAV939 administration in both KLF15KO and control mice, and their expression was significantly increased (SOX9, p < 0.0001 and p < 0.0001, respectively; Col2A1, p = 0.0003 and p < 0.0001, respectively). Wnt3A stimulation significantly increased the expression of β-catenin, Axin2, and ADAMTS5 in KLF15KO mice compared to control mice (p < 0.0001, p < 0.0001, and p < 0.0001, respectively). Conversely, the expression of SOX9 and Col2A1 was significantly decreased (p < 0.0001 and p = 0.0122, respectively). These findings were consistent with the in vivo results.

This study presents the first evidence that KLF15 negatively regulates Wnt/β-catenin signaling in cartilage and that it may play an important role in OA progression. Specifically, KLF15 suppresses β-catenin activity and helps to maintain cartilage matrix homeostasis in both mouse models and fetal costal chondrocytes in vitro.

Wnt ligand binding to Frizzled receptors and LRP5/6 inhibits β-catenin degradation, allowing it to accumulate in the cytoplasm and translocate into the nucleus, activating TCF/LEF-dependent transcription.^36,37 Excessive activation of the Wnt/β-catenin signaling pathway induces the expression of matrix-degrading enzymes, such as MMP13 and ADAMTS5, promoting OA pathology.³⁰ Experimental β-catenin overactivation in chondrocytes produces OA-like changes including osteophyte formation, cartilage thinning, and joint space narrowing. This suggests that β-catenin activation directly promotes cartilage destruction.³¹

Indeed, the nuclear localization of β-catenin and the expression of its target genes are increased in the articular cartilage of patients with OA.³⁸ In non-cartilage tissues, KLF15 has been shown to inhibit Wnt/β-catenin signaling by binding to β-catenin and promoting its proteasomal degradation.³⁹

KLF15 induction has also been shown to suppress Wnt/β-catenin signaling by binding to active β-catenin and inhibiting its phosphorylation in renal interstitial cells.⁴⁰ Furthermore, KLF15 may directly suppress β-catenin transcriptional activity by physically binding to β-catenin and forming a protein complex.

KLF15 deficiency enhanced β-catenin activity and increased expression of MMP13 and ADAMTS5 while reducing SOX9 and Col2A1, indicating a shift toward a catabolic state.⁴¹ These changes were further intensified by Wnt3a stimulation and partially rescued by XAV939, a β-catenin inhibitor that stabilizes AXIN2 and promotes β-catenin degradation. Consistent with our findings, previous studies have shown that β-catenin overactivation leads to structural abnormalities, not only in articular cartilage but also in intervertebral disc tissue, further supporting the destructive potential of this pathway in cartilage structure.⁴² XAV-939, a well-characterized small molecule inhibitor, stabilizes AXIN2 as a tankyrase inhibitor, thereby forming a degradation complex and promoting the phosphorylation-dependent degradation of β-catenin.⁴³ In a previous study, intra-articular injection of XAV939 in a DMM mouse model attenuated the severity of OA and demonstrated anti-catabolic effects in chondrocytes.⁴⁴

The results of this study indicate that KLF15 deficiency induces a catabolic environment via excessive activation of Wnt/β-catenin signaling, thereby promoting cartilage destruction. Furthermore, inhibition of the Wnt/β-catenin signaling pathway suppresses catabolic factor expression, indicating that OA-like changes associated with KLF15 deficiency occur via this pathway.

In addition, reduced anabolic factor expression in response to KLF15 deficiency indicates that the ability of chondrocytes to maintain differentiation and synthesize the matrix was significantly impaired. This reduction was partially restored by XAV939 administration, revealing that abnormal activation of the Wnt/β-catenin pathway is also involved in the breakdown of the anabolic environment. Therefore, KLF15 may suppress OA progression by interacting with and negatively regulating β-catenin in the cell nucleus, thereby suppressing the expression of cartilage-degrading enzymes, and maintaining an anabolic state (Fig. 6).

Limitations

This study has some limitations. First, KLF15 was specifically knocked out in cartilage tissue, so the environment may be different from the real-life disease environment. This is because OA is a complex disease that progresses with degeneration of synovium, bone, and meniscus in addition to cartilage tissue.^45,46 Second, fetal costal chondrocytes were used in vitro, not knee articular cartilage. Ideally, knee chondrocytes from adult mice should have been used. However, we were unable to obtain the estimated number of knee chondrocytes required for this experiment.

The findings of this study suggest that KLF15 is a potential therapeutic target for OA. If the Wnt/β-catenin pathway can be selectively suppressed by interventions that activate or restore KLF15 function, it may be possible to slow down or even prevent the destruction of articular cartilage and maintain cartilage differentiation. In addition, the development of small-molecule compounds that enhance the nuclear interaction between KLF15 and β-catenin and the restoration of KLF15 function by gene transfer should be considered as future approaches for treatment. However, because KLF15 is also involved in glucose metabolism and adipogenesis, its usefulness as a therapeutic target must be assessed in the context of its systemic effects.

Ethics approval and consent to participants

All animal experiments were approved by the Animal Experimentation Committee of Kobe University (Approval No. P230606) and conducted in accordance with institutional guidelines and the ARRIVE reporting standards. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing of interests

The authors declare no conflicts of interest.

Funding

This study was funded by the Grant-in-Aid for Scientific Research (21K09278).

Author’s contributions

Akira Saitoh (Acquisition of the data, Analysis and interpretation of the data, Drafting of the article, Final approval of the article), Shinya Hayashi (Conception and design, Analysis and interpretation of the data, Drafting of the article, Obtaining of funding, Final approval of the article), Toshiki Kitamura (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Takuma Hayashi (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Kohe Motono (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Shotaro Araki (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Takuma Maeda (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Yoshihito Suda (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Kensuke Wada (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Kemmei Ikuta (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Masanori Tsubosaka (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Tomoyuki Kamenaga (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Yuichi Kuroda (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Naoki Nakano (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Tomoyuki Matsumoto (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Tetsuya Hosooka (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Wataru Ogawa (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article), Ryosuke Kuroda (Analysis and interpretation of the data, Critical revision of the article for important intellectual content, Final approval of the article).

Acknowledgments

We wish to thank Ms. Minako Nagata, Ms. Maya Yasuda, and Ms. Kyoko Tanaka for their expert technical assistance. This study was funded by the Grant-in-Aid for Scientific Research (21K09278). We would like to thank Editage (www.editage.com) for English language editing.

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Deficiency of Krüppel-like factor 15 activates β-catenin and is involved in the development of osteoarthritis

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Abstract

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Background

Materials and methods

Generation of inducible cartilage-specific KLF15 knockout mice

Histological evaluation of cartilage degeneration

Immunohistochemistry

Isolation of mouse cartilage and chondrocytes

Co-immunoprecipitation

Double immunofluorescence

Cytoplasmic and nuclear protein extraction

Western blot analysis

Quantitative RT-PCR analysis

Statistical analyses

Results

KLF15 KO mice exhibit increased catabolic factor expression in cartilage

KLF15 KO mice exhibit decreased SOX9 expression, contributing to OA progression

Discussion

Limitations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1