Naproxen affects osteogenesis of human mesenchymal stem cells via regulation of Indian hedgehog signaling molecules.

Omar Salem,John Antoniou,Hong Wang,Insaf Hadjab,Abdulrahman M Alaseem,Fackson Mwale,Rahul Gawri,Ovidiu Ciobanu

doi:10.1186/ar4614

Abstract

IntroductionWe 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.MethodsMSCs 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.ResultsWhen 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.ConclusionsThese 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.

Highlights

We previously showed that type X collagen, a marker of late stage chondrocyte hypertrophy, 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
Alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OC) and collagen I (COL1A1) are important genes that define the osteogenesis of MSCs [24,25,26]
To test the effect of Npx on osteogenesis of MSCs, the expression of these genes was examined after MSCs were cultured in osteogenic differentiation medium with or without Npx for 3, 6 and 12 days

Summary

Introduction

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. 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 [1]. It is the major reason for seeking medical care and normal healing of injured tissues [4]. They are often inadequate and pain is the number one reason for undergoing joint replacement surgery [5]. Little is known about their effects on the subchondral bone repair in OA patients

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