Naproxen affects osteogenesis of human mesenchymal stem cells via regulation of Indian hedgehog signaling molecules
We previously showed that type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification), is constitutively expressed by mesenchymal stem cells (MSCs) from osteoarthritis patients and this may be related to Naproxen (Npx), a nonsteroidal anti-inflammatory drug used for therapy. Hedgehog (HH) signaling plays an important role during the development of bone. We tested the hypothesis that Npx affected osteogenic differentiation of human MSCs through the expression of Indian hedgehog (IHH), Patched-1 (PTC1) and GLI family members GLI1, GLI2, GLI3 in vitro.
MSCs were cultured in osteogenic differentiation medium without (control) or with 0.5 μM Npx. The expression of collagen type X, alpha 1 (COL10A1), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OC), collagen type I, alpha 1 (COL1A1) was analyzed with real-time reverse transcription (RT) PCR, and the ALP activity was measured. The osteogenesis of MSCs was monitored by mineral staining and quantification with alizarin red S. To examine whether Npx affects osteogenic differentiation through HH signaling, the effect of Npx on the expression of IHH, GLI1, GLI2, GLI3 and PTC1 was analyzed with real-time RT PCR. The effect of cyclopamine (Cpn), a HH signaling inhibitor, on the expression of COL10A1, ALP, OC and COL1A1 was also determined.
When MSCs were cultured in osteogenic differentiation medium, Npx supplementation led to a significant decrease in ALP gene expression as well as its activity, and had a tendency to decrease mineral deposition. It also decreased the expression of COL1A1 significantly. In contrast, the gene expression of COL10A1 and OPN were upregulated significantly by Npx. No significant effect was found on OC expression. The expression of IHH, PTC1, GLI1, and GLI2 was increased by Npx, while no significant difference was observed on GLI3 expression. Cpn reversed the effect of Npx on the expression of COL10A1, ALP, OPN and COL1A1.
These results indicate that Npx can affect gene expression during osteogenic differentiation of MSCs, and downregulate mineral deposition in the extracellular matrix through IHH signaling. Therefore, Npx could affect MSC-mediated repair of subchondral bone in OA patients.
KeywordsSubchondral Bone Osteogenic Differentiation Heterotopic Ossification Bone Repair Cyclopamine
collagen I, type 1
collagen X, type 1
Dulbecco’s modified Eagle’s medium
fetal bovine serum
GLI-Kruppel family member GLI
mesenchymal stem cell
non-steroidal anti-inflammatory drugs
polymerase chain reaction
Symptomatic osteoarthritis (OA), characterized by cartilage deterioration and osteophyte formation, is the most common joint disorder, affecting primarily the knees, hips, and hands, and the predominant symptom is pain . It is the major reason for seeking medical care and accounts for most of the use of non-steroidal anti-inflammatory drugs (NSAIDs) . Clinical recommendations for the sympathetic treatment of OA include acetaminophen (Acet) and NSAIDs such as ibuprofen (Ibu), diclofenac (Dic), naproxen (Npx), and celecoxib (Cele) . Unlike narcotics that target the central nervous system to alleviate pain, NSAIDs inhibit cyclooxygenase (COX) activity within the central nervous system and at the peripheral pain site to prevent the conversion of arachidonic acid into prostaglandins. Thus, NSAIDs can alter certain fundamental processes involved in the normal healing of injured tissues . However, they are often inadequate and pain is the number one reason for undergoing joint replacement surgery .
Subchondral bone sclerosis and progressive cartilage degradation are widely considered as the hallmarks of OA. Despite the increase in bone volume fraction, subchondral bone is hypomineralized due to abnormal bone remodeling [6, 7]. NSAIDs are used clinically to prevent ectopic bone formation that is also known as heterotopic ossification, and the efficacy of NSAIDs in the avoidance of heterotopic ossification has been documented in controlled clinical trials. Experimental studies also have documented the negative effects of NSAIDs on healing of skeletal tissues . In earlier work, it was found that NSAIDs suppressed proliferation and induced cell death in cultured osteoblasts . However, little is known about their effects on the subchondral bone repair in OA patients.
Mesenchymal stem cells (MSCs) are multipotent stromal cells capable of differentiating into multiple mesenchymal lineages, including osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic lineages, under different conditions [10, 11]. MSCs can differentiate to osteoblasts to form bone, while commitment of osteoprogenitor cells and differentiation into pre-osteoblasts, which eventually develop into mature osteoblasts, is a requisite . To reverse or retard the degeneration of articular cartilage and repair the subchondral bone, MSCs can be employed in biological therapy for OA .
In the previous study, we found that NSAIDs can affect the expression of both hypertrophic and osteogenic genes in MSCs during expansion, and naproxen (Npx) showed a stronger effect on gene expression in MSCs than other drugs . The effect of the drugs on gene expression in MSCs can influence the treatment of OA. Thus, it is important to understand whether Npx affects the osteogenesis of MSCs.
The Hedgehog (HH) protein family has been found in all vertebrates, since their original discovery in Drosophila. Indian hedgehog (IHH) regulates both chondrogenesis and endochondral bone formation [15, 16]. It is known to stimulate bone formation via a positive feedback loop. Disruption of HH signaling results in severe skeletal abnormalities . HH morphogens are secreted from the cells and bind to the receptor, Patched (PTC), to relinquish SMO from PTC suppression, thereby enabling activation of the GLI family of transcription factors, which are used as markers for HH signaling activity [18, 19]. They are responsible for HH-induced lineage commitment during MSC differentiation [20, 21].
In this research, the effect of Npx on hypertrophy and osteogenesis of MSCs was studied. Because IHH signaling can affect the osteogenesis of MSCs and other osteogenesis-related signaling pathways [21, 22, 23], the effect of Npx on IHH signaling was also studied to elucidate the mechanisms involved.
Materials and methods
Source and expansion of stem cells
Normal human MSCs were obtained from Lonza Walkersville Inc (Walkersville, MD, USA). According to the supplier, these cells were harvested from healthy human bone marrow and were positive for CD105, CD166, CD29 and CD44, but negative for CD14, CD34 and CD45. All cells were expanded in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin and were used within four passages. The culture medium and the supplement were from Wisent Inc (St-Bruno, QC, Canada).
In every well of a six-well plate (Sarstedt, QC, Canada), 5 × 105 MSCs were plated and cultured in expansion medium overnight. The floating cells were removed and the attached cells were cultured until the confluence was more than 90%. Then the cells were cultured in osteogenic differentiation medium for 3 days to allow the cells to adapt to the new environment. Afterwards, the cells were cultured in 3 mL/well of osteogenic differentiation medium with 0.5 μM Npx (Sigma-Aldrich, Oakville, ON, Canada). The cells cultured without Npx were used as control cells. To test the effect of cyclopamine (Cpn) on gene expression in MSCs, 0.5 μM cyclopamine (Sigma-Aldrich) was dissolved in the culture medium. The osteogenic differentiation medium was prepared with high-glucose DMEM containing 10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, 50 μM L-ascorbic acid, 100 units/mL penicillin, and 100 μg/mL streptomycin.
Total RNA isolation
After MSCs were cultured for 3, 6 and 12 days, they were washed with PBS and total RNA was extracted using Trizol reagent (Invitrogen, Burlington, ON, Canada) according to the protocol from the supplier [11, 14].
Reverse transcription and real-time PCR
Forward (1397–1416): CCACGTCTTCACATTTGGTG
Reverse (1573–1592): AGACTGCGCCTGGTAGTTGT
Forward (3982–4001): GAGAGCATGACCGATGGATT
Reverse (4140–4159): CCTTCTTGAGGTTGCCAGTC
Forward (1670–1690): AATGCCTGTGTCTGCTTTTAC
Reverse (1779–1799): ACAAGTAAAGATTCCAGTCCT
Forward (113–133): TGAAGGTCGGAGTCAACGGAT
Reverse (273–293): TTCTCAGCCTTGACGGTGCCA
Forward (676–695): AAGCGTGAGCCTGAATCTGT
Reverse (845–864): CAGCATGTACTGGGCTTTGA
Forward (199–218): CGACACCAGGAAGGAAGGTA
Reverse (382–401): TGCACAGAACGGAGGTAGTG
Forward (2285–2304): CTTTGCAAGCCAGGAGAAAC
Reverse (2428–2447): TTGTTGGACTGTGTGCCATT
Forward (519–538): CGGCTTTGACTGGGTGTATT
Reverse (718–737): AAAATGAGCACATCGCTGAA
Forward (20–39): TGAGAGCCCTCACACTCCTC
Reverse (170–151): CGCCTGGGTCTCTTCACTAC
Forward (759–778): TGAAACGAGTCAGCTGGATG
Reverse (920–901): TGAAATTCATGGCTGTGGAA
Forward (1460–1479): TCAGCAATGTCACAGCCTTC
Reverse (1688–1707): GTCGTGTGTGTCGGTGTAGG
Alkaline phosphatase activity
After MSCs were cultured in osteogenic differentiation medium with or without Npx for 3, 6, and 12 days, the cells were lysed and ALP activity was assayed with StemTAG™ Alkaline Phosphatase Activity Assay Kit (Colorimetric) (Cell Biolabs Inc, San Diego, CA, USA) according to the protocol from the supplier.
Mineralization analysis with alizarin red S
Osteogenic differentiation was monitored by mineral deposition with alizarin red S staining as previously described . To quantify the matrix mineralization, alizarin red S was extracted with 1 mL/well 100 mM cetylpyridinium chloride (Sigma-Aldrich) and measured at 570 nm.
Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Fisher’s protected least significant difference post hoc test, using Statview (SAS Institute, Inc). The results of three experiments with MSCs from three different donors were assessed, and the values were reported as mean ± SD. The significance was defined as a P-value <0.05.
If MSCs are to be used to stimulate bone repair in the presence of NSAIDs in the synovial fluid, it is essential that NSAIDs do not interfere with osteogenic differentiation. The results of the present work indicate that Npx can induce the expression of COL10A1 and OPN, and downregulate the expression of ALP and COL1A1 through activation of HH signaling. The downregulation of ALP gene expression led to the suppression of its activity and decreased mineral deposition. Osteogenesis occurs through an endochondral process involving cellular hypertrophy and mineralization in a manner analogous to the growth plate. Type X collagen is a marker of hypertrophy, whereas ALP, OC, OPN and COL1A1 are bone matrix proteins synthesized by osteoblasts [24, 25, 26]. Mineralization is a functional endpoint reflecting advanced osteogenesis. Achieving increased expression of OC, OPN, COL1A1, as well as ALP activity and mineralization is therefore an essential requisite for bone repair. Thus Npx has the potential to interfere with MSCs in repairing bone.
The effect of anti-inflammatory drugs on the osteogenesis of MSCs has been reported previously in other studies. It was reported that the osteogenic potential of MSC is inhibited/delayed by treatment with high-dose NSAIDs under inflammatory conditions, while no significant effects were observed in non-inflammatory-conditioned MSCs . In another experiment, it was reported that NSAIDs can inhibit bone formation via blockage of MSC chondrogenic differentiation - an important intermediate phase in normal endochondral bone formation - but not the osteogenesis of MSC . In our study, Npx had a tendency to downregulate matrix mineralization. Furthermore, its effect on the expression of OPN and COL1A1 could affect the function of bone. In addition to their effects on osteogenesis, high-dose and long-term administration of NSAIDs has been shown to induce adipogenesis in stem cells. For instance, Indomethacin can stimulate adipogenesis of MSCs . However, the effect of Npx on adipogenesis is not known. Interestingly, Npx was shown to have anti-obesity effects in animals when injected at a high dose .
A decrease in the expression of osteogenic markers and functional mineralization, and an increase in the expression of hypertrophic chondrocyte markers was observed after prolonged culture with Npx. During hypertrophy, cells enlarge in many tissues such as the growth plate, muscle and cartilage, which also occurs in conditions of degeneration, such as OA. However, we did not observe any appreciable increase in cell volume. It has been reported that calcium/calmodulin-dependent kinase II (CamkII) is an essential component of intracellular signaling pathways regulating chondrocyte maturation, and it can induce cell hypertrophy through a branched set of effector pathways including the transcriptional regulators Runx2 and Mef2c [33, 34]. Activation of CamkII activity can result in premature hypertrophic gene expression, with no cell swelling. Furthermore, chondrocytes can display morphological changes consistent with hypertrophy, without upregulating the expression of hypertrophic genes [33, 34]. Thus, the hypertrophic program may be more complex than previously thought.
Npx affected the expression of IHH, PTC1, GLI1 and GLI2 genes belonging to the HH signaling pathway, and affected the expression of osteogenic genes in MSCs at different time points. This might be the result of the interaction of different proteins involved in HH signaling . The upregulation of IHH, GLI1 and GLI2 can increase the HH signaling pathway positively, while the upregulation of PTC1 has a negative effect on the HH signaling function as more IHH molecules will interact with increased PTC1 and result in less SMO being released. As Npx did not affect the expression of GLI3, it is possible that GLI3 does not play a direct role in the osteogenesis of MSCs. Interestingly, we were unable to detect the expression of Sonic Hedgehog (SHH), another gene involved in HH signaling and osteogenesis of MSCs, with primers that were specific for human SHH.
The bone repair process in adults resembles normal development of the skeleton during embryogenesis. Numerous signaling pathways induce the osteogenic differentiation of MSCs. Although the mechanisms have not been fully discerned, and only HH signaling was studied in this research, other signaling pathways may also affect stem cell differentiation. As the results in this study indicate that Npx can affect treatment and lineage differentiation of MSC through the IHH signaling pathway, a better understanding of these underlying mechanisms and the effect of other NSAIDs on the osteogenesis of MSCs have far-reaching implications for improving bone repair for OA treatment.
The results support the concept that Npx has a dual role in that it can stimulate hypertrophic differentiation of MSCs, while suppressing osteogenic differentiation of MSCs and mineral deposition in the matrix through HH signaling.
This study was supported by Canadian Institutes of Health Research (CIHR).
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