Research ArticleExperimental Studies
Open Access

Ca2+/Calmodulin-dependent Protein Kinase II Inhibitor KN-93 Enhances Chondrogenesis of Bone Marrow Mesenchymal Stem Cells and Delays Chondrogenic Hypertrophy

KRITSARUT WUTTISIRIBOON, PATCHARAPORN TIPPAYAWAT, JUREERUT DADUANG and TEMDUANG LIMPAIBOON
In Vivo March 2023, 37 (2) 667-678; DOI: https://doi.org/10.21873/invivo.13127

Abstract

Background/Aim: Cartilage tissue engineering has been popularly applied in the treatment of articular cartilage defect because it is more effective in generating functional engineered cartilage than traditional methods. Although the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) is well established, it is often accompanied by undesired hypertrophy. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator in the ion channel pathway which is known to be involved in chondrogenic hypertrophy. Therefore, this study aimed to reduce the hypertrophy of BM-MSCs by inhibiting CaMKII activation. Materials and Methods: BM-MSCs were cultured in three-dimensional (3D) scaffold under chondrogenic induction with and without CaMKII inhibitor, KN-93. After cultivation, markers of chondrogenesis and hypertrophy were investigated. Results: KN-93 at a concentration of 2.0 μM had no effect on the viability of BM-MSCs, while the activation of CaMKII was suppressed. A long period of KN-93 treatment significantly up-regulated the expression of SRY-box transcription factor 9 and aggrecan on day 28 compared to untreated BM-MSCs. Furthermore, KN-93 treatment significantly down-regulated the expression of RUNX family transcription factor 2 and collagen type X alpha 1 chain on days 21 and 28. Immunohistochemistry showed increased expression of aggrecan and type II collagen while the expression of type X collagen was reduced. Conclusion: A CaMKII inhibitor, KN-93 is able to enhance chondrogenesis of BM-MSCs and suppress chondrogenic hypertrophy, suggesting its potential applicability in cartilage tissue engineering.

Key Words:

Articular cartilage is an avascular tissue composed of chondrocytes and extracellular matrix (ECM) containing mainly type II collagen, proteoglycans, and non-collagenous proteins (1). The functions of chondrocytes are the generation and maintenance of the ECM. Injured articular cartilage is difficult to restore because of its limited self-healing capacity (1). The current strategies are designed for treatment of articular cartilage defects such as microfractures, in total knee arthroplasty, and in autologous chondrocyte implantation (ACI) (2). Despite the ACI approach reducing the likelihood of immunological rejection, it has many drawbacks including donor-site morbidity, long recovery time, chondrocyte dedifferentiation, and fibrocartilage formation (3). Thus far, a number of procedures have been developed to eliminate the limitations of traditional methods. Combination of biomaterials, cell sources, and bioactive stimulators have been established to create functional engineered cartilage (4). A popular sourced cell type for cartilage regeneration is mesenchymal stem cells (MSCs) because of their extensive capacity for proliferation and differentiation without the risk of losing their phenotype (5). Successful chondrogenic induction of MSCs by growth factors such as transforming growth factor-β (TGF-β), bone morphogenic protein (BMP), fibroblast growth factor, and insulin-like growth factor in many types of fabricated scaffolds was reported (6). However, MSCs tend to acquire hypertrophy during chondrogenic induction, resulting in further development of endochondral bone formation. In fact, hypertrophic differentiation increases 10-fold in cell volume, which affects functional cartilaginous ECM synthesis (7).

Several studies reported the occurrence of MSC hypertrophy under the three-dimensional (3D) culture system. In detail, hypertrophic differentiation appeared after chondrogenic induction of bone marrow-derived (BM)-MSCs even though a BMP inhibitor was used to inhibit BM-MSC hypertrophy (8). Moreover, hypertrophy of MSC-derived chondrocytes was induced by chondrogenic medium supplemented with melatonin, resulting in the promotion of expression of hypertrophy-related genes and reduced type II collagen expression (9). Hypertrophic differentiation is identified by the expression of a series of hypertrophy markers such as RUNX family transcription factor 2 (RUNX2), type X collagen, alkaline phosphatase, and matrix metallopeptidase 13 (MMP13) (10). Hypertrophy markers have their individual functions in the process of cartilage mineralization. For instance, type X collagen and MMP13 expression in hypertrophic chondrocytes leads to the degradation of functional ECM and promotes calcification (11). In addition, the mechanisms of chondrogenic hypertrophy are widely studied in order to achieve greater understanding. It is well known that hypertrophic induction is associated with calcium (Ca2+) signaling pathways during the development of chondrocytes. In general, cytoplasmic Ca2+ is accumulated through extracellular Ca2+ influx and Ca2+ release from the endoplasmic reticulum. Cytoplasmic Ca2+ binds to calmodulin (CaM) to form a complex, which activates autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) after binding (12). Consequently, activated CaMKII derepresses chondrogenic hypertrophy by specifically phosphorylating histone deacetylase 4 (HDAC4). The binding of 14-3-3 protein to phosphorylated HDAC4 prevents nuclear import and promotes nuclear export of HDAC4, leading to suppression of its activity and transcriptional activation of genes involved in hypertrophy (13). Deacetylation of histone by HDAC4 results in DNA compaction within chromosomes, preventing the entry of hypertrophic transcription factor such as RUNX2 (14). The mechanism of the calcium signaling pathway in hypertrophy is depicted in Figure 1.

Figure 1.
Figure 1.

The mechanism of calcium signaling pathway in chondrogenic hypertrophy. Ca2+: Calcium; CaM: calmodulin; CaMKII: Ca2+/CaM-dependent protein kinase II; HDAC4: histone deacetylase 4; RUNX2: RUNX family transcription factor 2.

The strategic regulation of hypertrophic differentiation remains challenging for the application of MSCs in tissue engineering. According to the understanding of CaMKII function in chondrogenic hypertrophy, the suppression of its activity might reduce hypertrophic differentiation. 2-[N-(2-Hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocin amyl)-N-methylbenzylamine (KN-93) is the most widely used inhibitor of the cellular function of CaMKII (15). KN-93 was developed by Hidaka and co-workers to be more soluble than KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (16). Basically, KN-93 directly binds to the Ca2+/CaM complex and disrupts Ca2+/CaM binding activity in CaMKII activation (17). Therefore, this study hypothesized that the reduction of CaMKII activity by KN-93 might delay chondrogenic hypertrophy. Herein, we investigated the effects of KN-93 during the chondrogenic induction of BM-MSCs under a 3D culture system with stimulation by transforming growth factor-β3 (TGF-β3) and bone morphogenic protein 6 (BMP6).

Materials and Methods

Cell culture. Human BM-MSCs were obtained from Merck, Darmstadt, Germany and expanded to passage 4 in low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2.

Sulforhodamine B colorimetric assay (SRB). BM-MSCs at passage 4 were cultured at a density of 2×103 cells in a 96-well plate containing LG-DMEM with different concentrations of KN-93 (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with 5% CO2 for 5 days. LG-DMEM without KN-93 was used as an untreated control. After incubation, cells were fixed with 10% trichloroacetic acid at 4°C for 1 h. Thereafter, the fixed cells were gently washed with distilled water for five times and allowed to dry. SRB stain (0.057% in 1% acetic acid) was added and cells were incubated for 30 min at room temperature. Subsequently, the well plate was washed five times with 1% acetic acid to eliminate excess SRB stain and allowed to dry. Then, 10 mM Tris-base buffer was added and cells were incubated for 30 min. The absorbance was detected at 510 nm using a Sunrise microplate reader (Tegan, Zurich, Switzerland).

Protein extraction and western blot analysis. BM-MSCs at passage 4 were cultured in LG-DMEM containing 2.0 μM of KN-93 at 37°C with 5% CO2 for 21 days. The BM-MSCs were then trypsinized and washed with phosphate-buffered saline (PBS, pH 7.4) prior to protein extraction using CellLytic M reagent (Sigma-Aldrich) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The proteins were quantified using Coomassie Plus-The Better Bradford assay™ kit (Thermo Fisher Scientific). Protein samples (20 μg) were then separated on a 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis at 150 V for 1.30 h. Subsequently, the separated proteins were transferred to a polyvinylidene fluoride membrane using a semi-dry transfer unit (Amersham Biosciences, Amersham, UK). The membranes were soaked in 5% skimmed milk for 1 h at room temperature with gentle agitation to block non-specific binding, followed by incubation with primary antibodies: mouse monoclonal anti-CaMKII antibody (1:500; Abcam, Cambridge, UK), mouse monoclonal anti-pCaMKII (phosphor T286) antibody (1:2,000; Abcam), and rabbit polyclonal anti-actin (1:10,000; Abcam) at 4°C for 18 h. The membranes were then washed three times with Tris-buffered saline containing 0.1% Tween 20 for 10 min. Then 1:10,000 of horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam) or 1:2,000 donkey anti-rabbit IgG (Abcam) was added as secondary antibody and membranes were incubated for 1 h at room temperature. After washing, Enhanced Chemiluminescence Plus System (GE Healthcare, Chicago, IL, USA) was applied to the membranes and signals were visualized using an Amersham imager 600 (GE Healthcare). The band intensities were quantified by Image J software (U. S. National Institutes of Health, Bethesda, MD, USA).

Scaffold preparation and chondrogenic differentiation. The chondrogenic differentiation of BM-MSCs was induced within a scaffold fabricated from silk fibroin (SF), gelatin (G), chondroitin sulfate (C), hyaluronic acid (H), and aloe vera (A) (SF-GCH-A), a model for 3D culture. In brief, 3% (w/v) SF, 3% (w/v) GCH, and 0.35% (w/v) A solutions were blended at a ratio of 4:1:1 (v/v) before lyophilization. The SF-GCH-A scaffolds (3 mm diameter × 3 mm height) were sterilized with 70% ethanol under ultraviolet light for 1 h, rinsed three times with PBS (pH 7.4), and soaked in high-glucose Dulbecco’s modified Eagle’s medium (HG-DMEM; Thermo Fisher Scientific) for 4 h. The chondrogenic medium was prepared by supplementing HG-DMEM with 100 U/ml penicillin, 100 μg/ml streptomycin (all from Thermo Fisher Scientific), 50 μg/ml L-ascorbic acid-2-phosphate, 0.4 mM L-proline, 10−7 M dexamethasone, 1% Insulin-transferrin-sodium selenite, 250 ng/ml BMP6, and 10 ng/ml TGF-β3 (all from Sigma-Aldrich). Passage 4 of BM-MSCs at a density of 3×105 cells were seeded per scaffold and allowed to undergo initial cell attachment at 37°C with 5% CO2 for 4 h. After incubation, the BM-MSC-seeded constructs were transferred to a 24-well plate and cultured in chondrogenic medium at 37°C with 5% CO2 for 14 days to accelerate chondrogenic differentiation. Subsequently, the chondrogenic medium containing 2.0 μM of KN-93 was replaced and BM-MSCs were cultured for an additional 14 days. The chondrogenic medium was changed every 2 days up to 28 days. BM-MSCs cultured with chondrogenic medium without KN-93 were used as an untreated control. The BM-MSC-seeded constructs were prepared for gene expression and immunohistochemistry.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total RNA was extracted from BM-MSC-seeded constructs by TRIzol reagent (Thermo Fisher Scientific) and converted to cDNA using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s protocols. The expression of the following chondrogenesis- and hypertrophy-related genes was determined: Collagen type I alpha 1 chain (COL1A1), collagen type II alpha 1 chain (COL2A1), collagen type X alpha 1 chain (COL10A1), aggrecan (ACAN), matrix metallopeptidase 13 (MMP13), SRY-box transcription factor 9 (SOX9), and RUNX family transcription factor 2 (RUNX2). The housekeeping gene, ribosomal protein L13a (RPL13A), was used as an internal control. The primer sequences are shown in Table I. The qRT-PCR was performed on a Q3200 Real-Time PCR System (Bio-Gener, Hangzhou, PR China) using SYBR™ Green PCR Master Mix (Thermo Fisher Scientific). The relative mRNA expression was calculated by the 2–ΔΔCt method, where ΔCt=(Cttarget−Ctreference) (18).

View this table:
Table I.

List of primer sequences.

Immunohistochemistry. Paraffin-embedded scaffold sections at 4-μm thickness were deparaffinized and rehydrated. The deparaffinized sections were boiled in 0.01 M citrate buffer (pH 6.0) for 10 min to retrieve the antigen. The sections were soaked in 3% H2O2 for 1.30 h in a dark chamber to block endogenous peroxidase activity. After this procedure, the sections were washed with PBS (pH 7.4) containing 0.05% Tween 20. Fetal bovine serum at a concentration of 20% was added and the sections were incubated at room temperature for 2 h to block non-specific binding. Subsequently, the sections were incubated with 1:200 rabbit polyclonal anti-collagen II, 1:600 mouse monoclonal anti-collagen X, or 1:500 mouse monoclonal anti-aggrecan overnight at 4°C in a humidified chamber (all antibodies were purchased from Abcam). A section was incubated with antibody diluent instead of primary antibody as a negative control. After incubation, the sections were washed twice with PBS-Tween for 10 min and incubated with goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (Envision™ System; DAKO Corporation, Carpinteria, CA, USA) for 1.30 h at room temperature. The chromogen diaminobenzidine (DAKO Corporation) was added to the sections to visualize the antigen–antibody reaction, followed by nuclear counterstaining with Mayer’s hematoxylin.

Statistical analysis. Data are presented as the mean±standard deviation. All statistical analyses were performed using Prism8 (GraphPad Software, Boston, MA, USA). The differences among groups were analyzed using one-way analysis of variance with Tukey’s post-hoc comparisons test. The mean differences between two groups were analyzed using Student’s t-test. A value of p<0.05 was considered as statistically significant.

Results

Effect of KN-93 on viability of BM-MSCs. The cytotoxicity of KN-93 on BM-MSCs was determined by SRB assay. More than 90% of BM-MSCs were viable after treatment with KN-93 at different concentrations up to 2.0 μM (Figure 2A). By contrast, KN-93 at a concentration of 5.0 μM significantly reduced cell viability of BM-MSCs to 52.9±1.9% when compared to the untreated cells. The results indicated that the highest concentration of KN-93 applied while BM-MSCs remained viable was 2.0 μM. Thus, KN-93 at a concentration of 2.0 μM was chosen for further experiments.

Figure 2.
Figure 2.

The effect of KN-93 on the viability of human bone marrow-derived mesenchymal stem cells (A) and on Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation (B). Values are presented as the mean±standard deviation. **Statistically significant different at p<0.01.

KN-93 significantly reduces CaMKII phosphorylation. The effect of KN-93 on CaMKII phosphorylation was investigated by western blot analysis. The BM-MSCs were cultured with and without 2.0 μM KN-93. It was found that 2.0 μM KN-93 significantly reduced CaMKII phosphorylation (Figure 2B). The band intensity of phosphorylated CaMKII (pCaMKII) and of total CaMKII was normalized to that of the internal control, actin. The intensity of the phosphorylated form was then normalized to that of its total form. Remarkably, the normalized level of pCaMKII of KN-93 treated BM-MSCs was significantly lower than that of the untreated cells (Figure 2B).

KN-93 significantly enhances chondrogenesis and delays hypertrophy. The effect of KN-93 on chondrogenic and hypertrophic differentiation of BM-MSCs was investigated by monitoring related gene markers in BM-MSC-seeded constructs cultured in chondrogenic medium on days 7, 14, 21 and 28, to which 2.0 μM KN-93 was applied after chondrogenic induction for 14 days. The relative mRNA expression of individual genes at each specific time point was normalized to that of their respective genes on day 7. The chondrogenesis-related gene markers studied were COL1A1, SOX9, ACAN and COL2A1, while gene markers of hypertrophy were RUNX2, COL10A1 and MMP13. The effect of KN-93 on the expression of these genes is shown in Figure 3.

Figure 3.
Figure 3.

The effect of KN-93 on expression of chondrogenesis-related genes. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured in chondrogenic medium for 14 days, after which chondrogenic medium containing 2.0 μM of KN-93 was applied. The expression of chondrogenesis-related genes was determined by quantitative reverse transcription polymerase chain reaction in BM-MSCs without KN-93 from day 7 to day 28, and with KN-93 on day 21 and day 28. The relative mRNA expression was normalized to that of day 7. Values are expressed as the mean±standard deviation (n=6). Statistically significantly different at: *p<0.05 and **p<0.01 (between groups on the same day by t-test, among groups on different days by one-way analysis of variance). Statistically significantly different at p<0.05: #from day 7 and ‡from day 14. ACAN: Aggrecan; COL1A1: collagen type I alpha 1 chain; COL2A1: collagen type II alpha 1 chain; COL10A1: collagen type X alpha 1 chain; MMP13: matrix metallopeptidase 13; RUNX2: RUNX family transcription factor 2; SOX9: SRY-box transcription factor 9.

Expression of COL1A1, which was high in undifferentiated BM-MSCs, was dramatically reduced on day 14, and gradually decreased through days 21 and 28, indicating the success differentiation of BM-MSC; KN-93 had no effect on its expression.

In untreated BM-MSCs, the expression of SOX9, ACAN, and COL2A1 constantly increased from day 7 to day 21 but decreased on day 28. Indeed, the expression of SOX9 and ACAN was significantly increased on day 21 compared to day 7. The expression of COL2A1 was significantly increased on days 14, 21 and 28 compared to day 7, moreover, it was significantly increased on days 21 and 28 compared to day 14. Although expression of a series of chondrogenesis markers was increased from day 7 to day 21, the expression of SOX9 was significantly lower on day 28 compared to day 21, while no significant differences of ACAN and COL2A1 expression between days 21 and 28 were observed. On the other hand, KN-93, with a long period of chondrogenic induction, significantly enhanced the expression of SOX9 and ACAN, while delaying chondrogenesis at the beginning of KN-93 treatment. In detail, KN-93 treatment significantly down-regulated the expression of SOX9, ACAN, and COL2A1 on day 21 compared to the untreated cells. Remarkably, a long period of KN-93 treatment significantly up-regulated the expression of SOX9 and ACAN on day 28 compared to day 21. Moreover, the same effect was noted between treated and untreated cells on day 28. However, the extended KN-93 treatment had no effect on the expression of COL2A1.

In untreated cells, the expression of RUNX2 and COL10A1 on day 21 and day 28 was significantly higher than that on day 7 and day 14, while the expression of MMP13 did not significantly differ. This result indicates that hypertrophic differentiation occurred during chondrogenic induction. Interestingly, KN-93 treatment significantly down-regulated the expression of RUNX2 and COL10A1 on days 21 and 28 compared to the respective untreated cells. However, no significant difference of MMP13 expression after KN-93 treatment was observed. This finding indicates that KN-93 treatment significantly reduced the hypertrophic differentiation of BM-MSCs.

The expression of chondrocyte and hypertrophy-specific proteins such as aggrecan, type II collagen, and type X collagen was characterized by immunohistochemistry. Representative staining of a series of specific proteins is shown in Figure 4. The expression of type II collagen in KN-93-treated cells was slightly higher than that in the untreated cells (Figure 4A). Strikingly, strongly positive staining of aggrecan was observed in KN-93 treated BM-MSCs compared to untreated cells (Figure 4B). On the contrary, type X collagen expression in the KN-93 treated group was much lower than that in the untreated one (Figure 4C). The results for protein expression were in accordance with those for mRNA expression. This finding confirmed that KN-93 treatment enhanced chondrogenic differentiation and reduced hypertrophy of BM-MSCs.

Figure 4.
Figure 4.

Representative immunohistochemical staining of type II collagen (A), aggrecan (B), and type X collagen (C) of chondrogenic differentiated human bone marrow-derived mesenchymal stem cells with and without KN-93 treatment.

Discussion

The application of MSCs in cartilage tissue engineering requires the differentiation of MSCs into functional chondrocytes to manufacture cartilaginous ECM. The stability of chondrogenesis is the key to success in cartilage tissue engineering. However, undesired hypertrophy during the chondrogenic induction, which would result in apoptosis and ossification, is a major challenge for cartilage tissue engineering (19). Even though engineering cartilage tissue is a useful approach for osteoarthritis treatment, most current studies are still focusing on the potential use of MSCs in maintaining the chondrogenic phenotype during chondrogenic induction. In fact, hypertrophic differentiation induces several signaling pathways, which affects the functionality of sourced cells (10). Previous studies demonstrated that it is possible to suppress chondrogenic hypertrophy by several approaches such as co-culture of MSCs and articular chondrocytes (20), parathyroid hormone-related peptide treatment (21), and inhibitor of extracellular signal-regulated protein kinases 1 and 2 (22). To minimize the effect of hypertrophy, KN-93 was applied in this study. KN-93 treatment can affect cell survival if an inappropriate concentration is used. Previous studies used KN-93 at a concentration of 1.0 μM to block CaMKII pathway for studying the effect of Ca2+ on porcine BM-MSC adipogenesis (23) and the effect of melatonin on dopaminergic neuronal differentiation of amniotic fluid MSCs (24). Our study has shown that human BM-MSCs can survive under KN-93 treatment at concentrations up to 2.0 μM. A concentration of KN-93 was needed to have the most possible effect on inhibiting CaMKII activation without causing harm to BM-MSCs; we found that 2.0 μM of KN-93 effectively suppressed the phosphorylation of CaMKII in BM-MSCs.

Previous studies reported that TGF-β3 induced chondrogenic differentiation of MSCs in 3D culture (25, 26). BMPs have also been reported to induce in vitro chondrogenesis of human MSCs (27, 28). In our study, the chondrogenic differentiation of BM-MSCs in SF-GCH-A scaffolds was induced by TGF-β3 and BMP6. The cartilaginous ECM synthesis of MSCs was generated through chondrogenic induction (29). The investigation of chondrocyte-associated genes during chondrogenesis of MSCs elucidated the dynamics of chondrogenic differentiation. A previous study reported that cartilage-specific genes of MSCs were up-regulated after 14 days of chondrogenesis induction (30). It was reported that CaMKII is an important component of signaling pathways which regulate chondrocyte maturation (31). Moreover, a differentiation period of 14 days is recommended as a hypertrophic model of MSCs under standard chondrogenic culture conditions (32). To prevent effects of KN-93 on chondrogenic differentiation, we designed our study so that it would be added after 14 days of chondrogenic induction.

The down-regulation of COL1A1 expression indicated that the BM-MSCs underwent chondrogenic differentiation since COL1A1 was highly expressed in undifferentiated MSCs and its expression declined after differentiation (33). SOX9 is a crucial transcription factor in chondrogenesis that was reported to regulate the expression of type II collagen and aggrecan (34-36). The simultaneously progressive up-regulation of SOX9, ACAN, and COL2A1 indicated the chondrogenic differentiation of BM-MSCs, which declined at the late stage (day 28). Similarly, the BM-MSCs expressed high levels of glycosaminoglycan and type II collagen throughout the chondrogenic differentiation period, indicating the potential use of BM-MSCs under chondrogenic induction (37). By contrast, KN-93 down-regulated the expression of SOX9, COL2A1 and ACAN at the beginning of its period of treatment (day 21).

Ca2+/CaMKI was reported to regulate cell proliferation through cyclin-dependent kinase 4 (CDK4)/cyclin D, preventing CDK4/cyclin D assembly by KN-93, leading to G1 cell-cycle arrest (38). Accordingly, KN-93 treatment might delay chondrogenic differentiation on day 21 by cell-cycle arrest. Although KN-93 reduced the capacity of chondrogenesis at the early stage of the treatment, prolonged KN-93 treatment enhanced chondrogenesis with high SOX9 and ACAN expression on day 28. The mechanism of CaMKII in cell development has been widely explored. A previous study reported that the CaMKII inhibited cartilage ECM synthesis of BM-MSCs by influencing their differentiation (39). However, CaMKII inhibitor was found to enhance chondrogenesis in several studies. A study on CaMKII in chondrogenesis of BM-MSCs showed increased cartilage ECM generation and up-regulation of chondrogenesis-related genes in KN-93 treated BM-MSCs compared to untreated cells (39). Moreover, CaMKII was reported to be related to the non-canonical WNT pathway, which regulated chondrogenesis induced by WNT3A. The applied KN-93 was able to inhibit the non-canonical WNT pathway, resulting in increased COL2A1, ACAN, and SOX9 expression in MSCs infected with WNT3A lentivirus (40). Our findings suggest that KN-93 treatment enhanced chondrogenesis of BM-MSCs through the suppression of CaMKII activation. However, the increased SOX9 expression resulting from the KN-93 treatment was not able to induce COL2A1 expression on day 28. A previous study showed no positive correlation between SOX9 and COL2A1 expression in adult human articular chondrocytes. Moreover, no positive correlation between SOX9 and COL2A1 expression was observed in vitro after stimulating chondrocytes with interleukin-1 beta or with insulin-like growth factor 1, suggesting that SOX9 is not the key regulator of COL2A1, but may be involved in the maintenance of chondrocyte phenotype (41).

A high expression of hypertrophy markers including type X collagen, MMP13, and alkaline phosphatase during in vitro chondrogenic induction of MSCs has been found, indicating an unstable chondrogenic differentiation (7, 42). The hypertrophic differentiation of BM-MSCs was observed in our study, with constantly increased expression of RUNX2 and COL10A1 from day 21 to day 28. RUNX2 is well known as a transcription factor for chondrogenic hypertrophy and positively regulates type X collagen expression through BMP induction (43). In contrast, KN-93 treatment down-regulated RUNX2 and COL10A1 expression during chondrogenic differentiation. CaMKII is a positive inducer of chondrogenic hypertrophy because of its ability to suppress the functions of HDAC4 (44). The evidence showed that the chondrogenic hypertrophy was regulated by HDAC4 through the inhibition of RUNX2 activity (43). Thus, the CaMKII inhibitor KN-93 indirectly allowed the action of HDAC4, leading to the reduced expression of RUNX2. Moreover, the reduced COL10A1 expression was likely a result of the reduction in its upstream regulator, RUNX2.

Even though RUNX2 regulates the expression of MMP13, no significant difference in MMP13 expression was noted during chondrogenic differentiation. A previous study reported that hypoxia was necessary for endochondral ossification of hypertrophic chondrocytes through hypoxia-inducible factor 2α (45). Moreover, hypoxia-inducible factor 2α can trigger the expression of a series of MMPs and aggrecanase-1 resulting in articular cartilage destruction (46). In addition to RUNX2 activities, a low oxygen tension might be essential for increasing the expression of MMP13. Moreover, the study of Wang and coworkers demonstrated that increased RUNX2 expression in articular chondrocytes might be insufficient to increase MMP13 expression (47). Taken together, these data show that RUNX2 might not be a key regulator of MMP13 transcription since stable expression of MMP13 was observed during chondrogenic differentiation. In addition, it was reported that the cooperation of CaMKII and hes family bHLH transcription factor 1 modulates the development of osteoarthritis by inducing the expression of catabolic factors, such as MMP13 (48), suggesting the involvement of CaMKII in stimulating MMP13 expression. Although HDAC4 suppresses MMP13 transcription by interacting with and inhibiting RUNX2 DNA-binding activity (14), the results of our study revealed that treatment with KN-93 had no substantial impact on the expression of MMP13. This might be due to the low level of MMP13.

We elucidated the effects of KN-93 on BM-MSCs and the mechanism by which it enhanced chondrogenic induction of BM-MSCs and delayed hypertrophy, as depicted in Figure 5. In addition, the combination of the use of a 3D scaffold and KN-93 to suppress hypertrophy and promote chondrogenesis of BM-MSCs is advantageous in terms of cost-effectiveness, easy manipulation, and reduced processing time.

Figure 5.
Figure 5.

The mechanism of KN-93 action in chondrogenic differentiation. ACAN: Aggrecan; Ca2+: calcium; CaM: calmodulin; CaMKII: Ca2+/CaM-dependent protein kinase II; COL2A1: collagen type II alpha 1 chain; COL10A1: collagen type X alpha 1 chain; HDAC4: histone deacetylase 4; HIF-2α: hypoxia-inducible factor 2 alpha; MMP13: matrix metallopeptidase 13; RUNX2: RUNX family transcription factor 2; SOX9: SRY-box transcription factor 9.

In conclusion, the successful chondrogenic differentiation of BM-MSCs with high expression of chondrogenesis markers was demonstrated. CaMKII inhibitor, KN-93, at the optimal concentration of 2.0 μM, suppresses the activation of CaMKII with no effect on BM-MSC viability. Moreover, KN-93 treatment enhances chondrogenesis and delays chondrogenic hypertrophy of BM-MSCs. Therefore, KN-93 is a good alternative agent for efficiently improving in vitro chondrogenesis of BM-MSCs in cartilage tissue engineering.

Acknowledgements

This study was financially supported by the Thailand Science Research and Innovation (TSRI) through the Royal Golden Jubilee Ph.D. Program (grant no. PHD/0049/2558 to Kritsarut Wuttisiriboon), and the Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand. We appreciate Srinagarind Hospital Excellence Laboratory, Srinagarind Hospital, Faculty of Medicine, Khon Kean University, Khon Kaen, Thailand for facilitating gene expression analysis via real-time PCR. We would like to specially thank the Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand for immunohistochemistry slide preparation.

Footnotes

  • Authors’ Contributions

    Temduang Limpaiboon: Funding acquisition, project administration, supervision, conceptualization, methodology, validation, writing - review and editing. Kritsarut Wuttisiriboon: Conceptualization, methodology, investigation, formal analysis, validation, and writing - original draft. Patcharaporn Tippayawat: Conceptualization, methodology and validation. Jureerut Daduang: Conceptualization, methodology and validation.

  • Conflicts of Interest

    The Authors report no conflicts of interest.

  • Received January 17, 2023.
  • Revision received February 4, 2023.
  • Accepted February 9, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Ca2+/Calmodulin-dependent Protein Kinase II Inhibitor KN-93 Enhances Chondrogenesis of Bone Marrow Mesenchymal Stem Cells and Delays Chondrogenic Hypertrophy
KRITSARUT WUTTISIRIBOON, PATCHARAPORN TIPPAYAWAT, JUREERUT DADUANG, TEMDUANG LIMPAIBOON
In Vivo Mar 2023, 37 (2) 667-678; DOI: 10.21873/invivo.13127
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