title: Naproxen affects osteogenesis of human mesenchymal stem cells via regulation of Indian hedgehog signaling molecules.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Omar
authors.initials: O
authors.lastname: Salem
authors.forename: Hong Tian
authors.initials: HT
authors.lastname: Wang
authors.forename: Abdulrahman M
authors.initials: AM
authors.lastname: Alaseem
authors.forename: Ovidiu
authors.initials: O
authors.lastname: Ciobanu
authors.forename: Insaf
authors.initials: I
authors.lastname: Hadjab
authors.forename: Rahul
authors.initials: R
authors.lastname: Gawri
authors.forename: John
authors.initials: J
authors.lastname: Antoniou
authors.forename: Fackson
authors.initials: F
authors.lastname: Mwale
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000009:adverse effects*; D002454:Cell Differentiation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009288:Naproxen / Q000009:adverse effects*; D010012:Osteogenesis / Q000187:drug effects*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D053823:Hedgehog Proteins; C509246:IHH protein, human; D009288:Naproxen