Functional differences of Toll‐like receptor 4 in osteogenesis, adipogenesis and chondrogenesis in human bone marrow‐derived mesenchymal stem cells

Abstract Multipotent human bone marrow‐derived mesenchymal stem cells (hMSCs) are promising candidates for bone and cartilage regeneration. Toll‐like receptor 4 (TLR4) is expressed by hMSCs and is a receptor for both exogenous and endogenous danger signals. TLRs have been shown to possess functional differences based on the species (human or mouse) they are isolated from therefore, the effects of knockdown of TLR4 were evaluated in humans during the differentiation of MSCs into bone, fat and chondrocyte cells in vitro. We investigated the expression profile of TLR4 during the differentiation of hMSCs into three different lineages on days 7, 14 and 21 and assessed the differentiation potential of the cells in the presence of lipopolysaccharide (LPS, as an exogenous agonist) and fibronectin fragment III‐1c (FnIII‐1c, as an endogenous agonist). TLR4 expression increased following the induction of hMSC differentiation into all three lineages. Alkaline phosphatase activity revealed that FnIII‐1c accelerated calcium deposition on day 7, whereas LPS increased calcium deposition on day 14. Chondrogenesis increased in the presence of LPS; however, FnIII‐1c acted as a reducer in the late stage. TLR4 silencing led to decreased osteogenesis and increased adipogenesis. Furthermore, Wnt5a expression was inversely related to chondrogenesis during the late stage of differentiation. We suggest that understanding the functionality of TLR4 (in the presence of pathogen or stress signal) during the differentiation of hMSCs into three lineages would be useful for MSC‐based treatments.


| INTRODUC TI ON
Multipotent adult mesenchymal stem cells (MSCs) are adherent, fibroblast-like and non-hematopoietic cells, that have the capability to self-renew and differentiate into three lineages including osteoblasts, chondrocytes and adipocytes. 1,2 It is well known that MSCs have a role in the regulation of proliferation, migration, differentiation and immunomodulation of cells. 3,4 It is necessary to understand how these functions are regulated by MSCs. The expression of TLRs was observed on different types of cells. 5,6 TLRs are also expressed on stem/progenitor cells and have been shown to control the survival, proliferation, differentiation and migration of these cells. 7 Therefore, it is possible that MSCs perform many of their functions through TLRs. [8][9][10][11] TLRs are type-I transmembrane glycoproteins comprising two domains, the leucine-rich repeat (LRP) domain that can identify pathogen-associated molecular patterns (PAMPs) and the Toll/IL-1 receptor (TIR) domain that activates adaptor molecules including myeloid differentiation primary response 88 (MYD88), TIR domain receptor-associated protein (TIRAP), TRIF-related adaptor molecule (TRAM) and TIR domain-containing adaptor protein inducing INFβ (TRIF). 12 Finally, nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) are activated that enhance the production of proinflammatory cytokines. 13 In addition to PAMPs (such as lipopolysaccharides), 13 TLRs are also activated in the presence of heat-shock proteins (HSPs), S100 proteins and extracellular matrix fragments that named damageassociated molecular patterns (DAMPs) (such as fibronectin).
DAMPs are released upon alteration in the structures of tissues, cellular stress or tissue damage. [14][15][16] The FnIII-1c is a Type III domain of fibronectin that, as a component of extracellular matrix molecules (ECM) can activate TLR4 signalling. Mechanical injury and tissue damage could drive the fate of hMSCs via TLRs. 17 TLRs when triggered have a regulatory role in immune regulation, migration and the recruitment of MSCs in damaged tissues. 10 We investigated how the presence of pathogens and stress/danger signals affects the signalling pathway of TLR4 during the differentiation of hMSCs into three different lineages. The role of Wnt5a in response to inflammation 18 and differentiation 19 in MSCs has been shown previously.
The Wnt5a acts via the non-canonical pathway and is involved in osteogenesis. 20 The WNT5A/FZD4/JNK pathway is effective in inducing osteogenesis in mouse bone marrow (BM)-derived MSCs via mechanical loading. 21 Recently, it has been shown that the release of FnIII-1c is controlled by mechanical forces and proteolysis. 17 It was recently clarified that TLR4 performed its role in osteogenesis via Wnt5a in mouse BM-MSCs, 22 but its role in humans is not known. Studies have not yet determined a relationship between the pattern of TLR4 expression (in the presence and absence of endogenous and exogenous ligands) and the pattern of gene expression during differentiation of MSCs into fat and cartilage cells. Moreover, the effect of FnIII-1c as a stress signal on the differentiation potential of human MSC into bone, fat and chondrocyte cells is unknown.
In the present study, we tried to elucidate the molecular mechanisms and functions of TLR4 during the differentiation of human MSCs into three lineages. First, we studied expression patterns of TLR4 during differentiation into the three lineages and then created a model for inducing pathogen-related and tissue damagerelated stress conditions through the activation of TLR4 via LPS and FnIII-1c, respectively. Second, the expression of genes during differentiation of MSCs into all three lineages in the presence of both ligands was investigated. Given that so far there is no report on the effect of TLR4 knockdown on the differentiation capabilities of human MSCs into any of the three lineages, the function of TLR4 was evaluated by its knockdown in MSCs during differentiation into the respective lineages. In addition, we also investigated whether the release of FnIII-1c due to mechanical forces and proteolysis can affect the differentiation of human MSCs via Wnt5a expression.
We examined the involvement of Wnt5a in the differentiation induced by TLR4.

| Alizarin red quantification
Alizarin red quantification was used for the evaluation of mineralization of the cells on exposure to FnIII-1c at the following concentrations: 0.5, 1 and 1.5 µg/mL. First, the cells were fixed with 4% paraformaldehyde, and then Alizarin red was used for staining.
Alizarin red quantification (Sigma-Aldrich, Catalog No. ECM815) was performed according to the manufacturer's instructions by measuring absorbance at 405 nm in a microplate reader.

| Alkaline phosphatase (ALP) activity
The effects of TLR4 ligands and TLR4 knockdown on the differentiation of BM-MSCs into osteoblast cells were investigated as a function of ALP activity after 7, 14 and 21 days. An alkaline phosphatase assay kit (Colorimetric, Abcam, Catalog No. ab83369) was used to measure activity according to the manufacturer's protocol. The BCA protein (Novagen, Catalog No. 71285-3) assay kit was used to determine the protein concentration. The specific activity was determined through enzyme activity normalized by total protein content.

| RNA interference
The cells were seeded at a density of 2 × 10 5 cells in 6-well plates.
siRNA-induced silencing of TLR4 gene was determined by Real-time PCR and western blot techniques.

| Real-time PCR
To study the expression levels of genes involved in osteogenic, adipogenic and chondrogenic differentiation (For more details, see Table S1) as well as Wnt5a and TLR4, total RNA was ex-  Table S1, the sequence of the specific primers for each gene is listed. No. RPN2232) and proteins were visualized by the chemiluminescence imaging system (UVITEC Cambridge).

| Statistical analysis
Data were analysed using Prism statistical software (version 8). The statistical significance of the differences between groups was determined by one-way analysis of variance (ANOVA) and followed by post hoc Tukey test. Also, the comparison between the two groups was done with t-test. A P-value of .05 was considered significant.

| TLR4 expression increased during osteogenic, chondrogenic and adipogenic differentiation of hMSCs
To determine whether osteogenic, chondrogenic and adipogenic differentiation can affect TLR4 expression, human BM-MSCs were cultured and differentiated into three different lineages using induction media for 7, 14 and 21 days. The cells also were treated with 1 µg/mL LPS, this concentration was selected based on a previous study. 23,24 To determine whether FnIII-1c affects the osteogenic and adipogenic differentiation of MSCs, we added FnIII-1c in an appro- During chondrogenic differentiation, TLR4 expression on treatment with FnIII-1c was significantly increased from day 14 ( Figure 2C).

| Both LPS and FnIII-1c activation promoted osteogenic differentiation of hMSCs
To detect whether TLR4 affects osteogenic differentiation differently upon exposure to its ligands, hMSCs were cultured in the and FnIII-1c showed a staining density lower than that of the untreated group ( Figure 4D). F I G U R E 2 TLR4 expression during the differentiation of MSCs into the three respective lineages in the presence and absence of LPS or FnIII-1c. MSCs were cultured for differentiation into three lineages then the cells were treated with and without 1 µg/mL LPS or FnIII-1c for 7, 14 and 21 days. The mRNA levels of TLR4 were detected in cells cultured in (A) osteogenic, (B) adipogenic, and (C) chondrogenic media using real-time PCR. Data are presented as mean ± SEM of ratios relative to β-actin levels (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001 F I G U R E 3 Effect of LPS and FnIII-1c activation on the expression of genes involved in osteogenic differentiation of hMSCs. MSCs were cultured under osteogenic medium with and without 1 µg/mL LPS or FnIII-1c for 7, 14 and 21 d for all the experiments mentioned below. The mRNA levels of (A) Runt-related transcription factor 2, (B) osteocalcin, and (C), bone morphogenetic protein-2 were evaluated using real-time PCR. (D) ALP activity measurement and (E) characterization of hMSCs were conducted using Alizarin red staining (scale bar = 200 mm), MSCs were cultured in DMEM medium in the control group (Con). Data are presented as mean ± SEM of ratios relative to β-actin levels (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001

| TLR4 activation by LPS but not FnIII-1c enhanced chondrogenic differentiation of hMSCs
To verify whether TLR4 endogenous and exogenous ligands affect the chondrogenic differentiation of hMSCs, LPS and FnIII-1c were added to the chondrogenic medium. LPS stimulated the mRNA expression of SOX9 and COL2A1 compared to the untreated group on day 21 but FnIII-1c activation strongly decreased expression of both SOX9 and COL2A1 compared to the untreated differentiated group ( Figure 5A,B). After 21 days, the pellets that were cultured with and without LPS or FnIII-1c were assessed for the chondrogenic phenotype using toluidine blue and Safranin-O staining, the LPS and untreated groups had acquired a clear phenotype of differentiation in comparison with the FnIII-1c group ( Figure 5D,E).  Figure 7I). ALP activity in the untreated group reached its maximum on day 14. The highest peak of ALP activity in the FnIII-1c group was achieved on day 7. It was also observed that the TLR4 knockdown caused a decrease in ALP activity. Though ALP activity increased on treatment with LPS on day 7, the highest peak of activity was observed on day 14, and the TLR4 knockdown caused a decrease in activity here as well ( Figure 7J).

| LPS promoted Wnt5a expression during osteogenesis in a stage-dependent manner and FnIII-1c inhibited Wnt5a expression during adipogenesis
We analysed whether stress-induced ligand (FnIII-1c) can stimulate MSC differentiation into the three respective lineages in comparison with pathogen ligand (LPS). During osteogenic differentiation, Wnt5a expression increased in a time-dependent manner, and LPS treatment significantly enhanced the Wnt5a level on day 7 (P < .0001) ( Figure S1). Wnt5a expression upon FnIII-1c activation significantly decreased during osteogenesis and adipogenesis on days 7, 14 and 21 in comparison with the untreated group ( Figure S1). During chondrogenic differentiation, Wnt5a expression on treatment with and without LPS or FnIII-1c reached a maximum on day 14 ( Figure S1).

During osteogenesis, Wnt5a expression was decreased in the TLR4
knockdown group in all three time-points compared to in the control group, however, this difference was not significant ( Figure S1). After knockdown of TLR4, there was no significant difference in Wnt5a levels between the control and TLR4 knockdown group upon adipogenesis induction ( Figure S1). However, during chondrogenesis, upon TLR4 knockdown, Wnt5a expression was significantly higher than in the control group on day 21 (Due to differences between tested groups during chondrogenesis on day 21, the TLR4 knockdown effects were studied at this time-point) ( Figure S1). Transfer of LPS to the TLR4/MD2 complex is facilitated by LBP and CD14, but this transition of LPS to the molecular complex is a dynamic process required for the activation of the immune system. 25 Therefore, this dynamic process may also affect the potential for the differentiation of the MSCs. In dermal fibroblasts, it has been shown that differences in the response of TLR4 to Tenascin C, FnIII-1c or FnEDA may be due to differences in the co-receptor that stimulate it. Therefore, the heterogeneity of the TLR4 receptor complex varies as a function of TLR4 activation. 17 TLRs regulate many functions of MSCs upon stimulation by their ligands. 26 LPS treatment did not affect the properties of immunosuppression and immunogenicity in human adiposederived stem cells (hASCs). 27 Proliferation in mouse BM-MSCs was enhanced following TLR4 stimulation. 28 Recently, it has been shown that different responses occur on stimulation of TLR depending on the species (human or mouse) as well as the cell type of MSCs. 9,29,30 Therefore, we evaluated whether a pattern of TLR4 expression is Data are presented as mean ± SEM of ratios relative to β-actin levels (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001

| D ISCUSS I ON
Our results showed that chondrogenesis was increased by LPS In hMSCs, after stimulation by LPS, expression of IL-1β, IL-6 and tumour necrosis factor (TNF)-a was enhanced but treatment with fibronectin led to an increase in the production of matrix metalloproteinase-3 (MMP3). 10 Production of IL-6 was increased after a bone fracture in the inflammatory phases of fracture healing. 38 In another study, LPS in addition to producing inflammatory cytokines, increased IP10 (CXCL10) expression that was activated via IFN1β, indicating that LPS has affected both MyD88-dependent and MyD88independent pathways of hASCs. 39 The MMPs play a role in ECM degradation, during chondrogenesis the levels of proteoglycans increased, which was associated with increased levels of genes related to ECM and decreased levels of MMP3. 40 Overall it seems the pro- The molecular mechanism of TLR4 in the differentiation of hMSCs into three lineages is still unknown. However, the MAPK pathway also plays a role in the osteogenesis of MSCs. 41 Maximum ERK activation was observed during osteogenesis of hADSCs at day 7 which LPS stimulation enhanced. 32 Stimulation of both LPS and fibronectin triggered the phosphoinositide 3-kinase (PI3K) pathway, upstream of the MAPK and NF-κB pathways in hMSCs. 10 Recently, it has been shown that FnIII-1c activates p38 MAP kinase and stable mRNA IL-8. 17 Therefore, it suggests that fibronectin may exert its effect on osteogenesis via MAPK.
Another pathway involved in the differentiation of MSCs is the Wnt5a pathway. TLRs and NF-kB pathways play a role in Wnt5a expression. 42 In human dental pulp stem cells, Wnt5a expression increased due to LPS via the pathways of TLR4/MyD88, NF-kB or PI3K/AKT. 43 It has been shown that TLRs alone are not enough for the regulation of the expression of Wnt5a but it was suggested that particular requirements for cell type and condition of differentiation could also affect. 42 These results raise the possibility that TLR4 may play a diverse role in regulating Wnt signalling in different tissues. Overexpression of Wnt5a via the promoter of osteopontin drives osteogenesis in MSCs and suppressing Wnt5a promoted differentiation of MSCs into pre-adipocytes. 44 In our study, up-regulation of Wnt5a expression was observed during osteogenesis and adipogenesis and it seems that Wnt5a stimulation by LPS increases osteogenesis on day 7.  45 In the stages of early chondrogenesis, F I G U R E 6 TLR4 silencing by psiTLR transfection. MSCs were transfected with psiTLR4 and psiRNA-LucGL3 (control). (A) TLR4 levels in the TLR4 knockdown and control groups were detected through western blotting. MSCs were transfected with psiTLR4, transfection of hMSCs with psiTLR4 was confirmed using (B) fluorescence microscope, and (C) light microscope (Scale bar = 200 mm). (D) merged state is shown. TLR4 expression after knockdown of TLR4 in MSCs during (E) osteogenic, (F) adipogenic, and (G) chondrogenic differentiation was detected using real-time PCR. Data are presented as mean ± SEM of ratios relative to β-actin levels (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001 F I G U R E 7 Effect of TLR4 knockdown on the expression of genes involved in osteogenic, adipogenic and chondrogenic differentiation. MSCs were transfected with psiRNA-hTLR4 (SiTLR4 group) and psiRNA-LucGL3 (control group). The mRNA levels of (A) Runt-related transcription factor 2, (B) osteocalcin, (C) bone morphogenetic protein-2, (D) lipoprotein lipase, (E) Fatty acid-binding protein 4 (FABP4), (F) peroxisome proliferator-activated receptor gamma (PPARγ), (G) SOX9, (H) type II collagen (COL2A1) and (I) Aggrecan (ACAN) were evaluated using real-time PCR for (A-C) osteogenic (D-F) adipogenic and (G-I) chondrogenic differentiation, respectively. (J) ALP activity of treated groups undergoing osteogenic differentiation was evaluated, MSCs were cultured in DMEM medium in the control group (Con). Data are presented as mean ± SEM of ratios relative to β-actin levels (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001 Wnt5a suppresses the canonical pathway for differentiation into cartilage, however, it induces the non-canonical pathway which contributes to the destruction of cartilage. 46 Therefore, it seems that Wnt5a plays a role in the onset of chondrogenic differentiation. To determine definitively the effect of Wnt5a on the differentiation of mesenchymal stem cells, the effect of Wnt5a siRNA on the differentiation of hMSCs in the absence and presence of TLR4 agonists should be determined.

| CON CLUS ION
We observed that TLR4 expression increased during the differentiation of MSCs into all three lineages. However, the basic expression of TLR4 is the mechanism that preserves the self-renewal capacity of MSCs. We also revealed that TLR4 knockdown suppresses osteogenesis while promoting adipogenesis in human MSCs. Our data clarify that the FnIII-1c promotes osteogenesis in the early stages of differentiation but it decreased chondrogenesis in the late stage of the process.
Up-regulation of TLR4 has a direct link with enhanced osteogenesis and chondrogenesis depending on the type of ligand and the stage of differentiation but its expression was inversely related to adipogenesis. This study elucidates how the presence of stress or pathogens alters TLR4 expression and if the differentiation potential of MSCs is affected, which is important to comprehend from a clinical or treatment perspective.

ACK N OWLED G EM ENTS
The authors are thankful to the Vice Chancellor of Research of Qazvin University of Medical Sciences and Royan Institute for their support.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are available from the corresponding author.