Osteoarthritis Cartilage. 2014 Sep;22(9):1327-36. doi: 10.1016/j.joca.2014.07.009.

Clodronate exerts an anabolic effect on articular chondrocytes mediated through the purinergic receptor pathway.

 

Rosa RG1, Collavino K2, Lakhani A3, Delve E4, Weber JF5, Rosenthal AK6, Waldman SD7.
  • 1Human Mobility Research Centre, Kingston General Hospital, Kingston, Ontario, Canada. Electronic address: renata.giardini@gmail.com.
  • 2Department of Chemical Engineering, Ryerson University, Toronto, Ontario, Canada; Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Ontario, Canada. Electronic address: kristina.collavino@ryerson.ca.
  • 3Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada. Electronic address: anand.lakhani@queensu.ca.
  • 4Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada. Electronic address: elizabeth.delve@mail.utoronto.ca.
  • 5Department of Mechanical & Materials Engineering, Queen’s University, Kingston, Ontario, Canada. Electronic address: weberj@me.queensu.ca.
  • 6Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, and the Zablocki VA Medical Center, Milwaukee, WI, USA. Electronic address: ann.rosenthal@va.gov.
  • 7Department of Chemical Engineering, Ryerson University, Toronto, Ontario, Canada; Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Ontario, Canada. Electronic address: swaldman@ryerson.ca.

 

Abstract

OBJECTIVE: Bisphosphonates are commonly used anti-osteoporotic drugs which have controversial effects on joint diseases including osteoarthritis. Certain bisphosphonates have been shown to have anabolic effects on cartilage which could have important ramifications for their proposed effects in vivo; however, the underlying mechanisms are poorly understood. Thus, the purpose of this study was to characterize the effects of clodronate on primary articular chondrocyte metabolism and to determine the underlying signaling pathways responsible.

DESIGN: The effects of clodronate and pamidronate on extracellular matrix (ECM) biosynthesis, accumulation and MMP-13 activity were observed in high density, 3D cultures of bovine articular chondrocytes for up to 4 weeks were evaluated. Mechanisms were delineated by measuring intracellular Ca(2+) signaling and the effects of pharmacologic inhibition of the purinergic receptor pathway.

RESULTS: Clodronate (100 μM) induced an anabolic effect (increased biosynthesis by 13-14%) which resulted in an 89-90% increase in ECM accumulation after 4 weeks of culture and without an associated effect on matrix turn-over. Stimulation by clodronate resulted in a 3.3-fold increase in Ca(2+) signaling and pharmacological inhibitor experiments suggested that the anabolic effects exerted by clodronate are transduced through the purinergic receptor pathway.

CONCLUSIONS: These findings support the previous notion that certain bisphosphonates may be useful as adjunctive therapies to potentially ameliorate progression of cartilage degeneration and improve arthritis management.

Copyright © 2014 Osteoarthritis Research Society International.

KEYWORDS: Anabolism; Bisphosphonates; Calcium signaling; Chondrocytes; Clodronate; Purinergic signaling

PMID: 25042551

 

Supplementary Information

Bisphosphonates are commonly used anti-osteoporotic drugs that exert both physiochemical (mineralization inhibition) and biological (osteoclast apoptosis) effects making them well suited to inhibit bone resorption. Interestingly, these molecules have also been ascribed anti-inflammatory, chondro-protective, and anti-catabolic effects, suggesting that they may be promising drugs for the treatment of osteoarthritis and rheumatoid arthritis. Although certain bisphosphonates (e.g. etidronate, clodronate) have been shown to elicit anabolic effects on cartilage, recent clinical studies have generally been inconclusive.

While the physiochemical effects of bisphosphonates on mineralization are well known, their molecular mechanisms on cellular function have been broadly categorized into two groups based on bisphosphonate structure. After cellular uptake, nitrogen-containing bisphosphonates (e.g. pamidronate, zolendronate) interfere with the mevalonate biosynthetic pathway leading to inhibition of bone resorption by disrupting both osteoclast function and survival. Alternatively, non-nitrogen-containing bisphosphonates (e.g. etidronate, clodronate) appear to be metabolized into the non-hydrolyzable ATP analog adenosine 5′(β,γ‑dichloromethylene) triphosphate (AppCCl2p) which inhibits mitochondrial metabolism resulting in osteoclast apoptosis. Interestingly, however, chondrocytes utilize ATP as an autocrine/paracrine signaling molecule during mechanical loading transduced through the purinergic receptor pathway. Due to the structural similarity between ATP and the non-hydrolyzable bisphosphonate metabolite AppCCl2p, we hypothesized that non-nitrogen containing bisphosphonates elicit an anabolic response in chondrocytes through the extracellular transport of AppCC2p and its interactions with the purinergic receptor pathway.

Studies were then conducted on isolated chondrocytes stimulated with non-nitrogen containing (clodronate) as well as nitrogen-containing (pamidronate) bisphosphonates to assess their effects on extracellular matrix synthesis, matrix turn over, and intracellular calcium signaling. Results indicated that clodronate, and not pamidronate, induced an anabolic effect and that this response appeared to be mediated by the export of an intracellular ATP-analog which then signals through the purinergic receptor pathway. From these results, we proposed the following signaling pathway (Figure 1). After cellular uptake by fluid phase endocytosis (pinocytosis), clodronate is metabolized into the non-hydrolyzable ATP analog adenosine 5′(β,γ‑dichloromethylene) triphosphate (AppCCl2p) by aminoacyl-tRNA synthetases. This metabolite is then released into the extracellular space initially through the connexin-43 hemi-channel. Further AppCCl2p release occurs as a result of P2X7 and/or P2X4 receptor binding leading to additional release through its known association with the ATP release channel, pannexin 1. Extracellular AppCCl2p then binds to P2Y receptors (most likely P2Y2) resulting in the stimulation of extracellular matrix synthesis. These findings support the previous notion that certain bisphosphonates (i.e. non-nitrogen containing bisphosphonates) may be useful as adjunctive therapies to potentially ameliorate progression of cartilage degeneration and improve arthritis management.

 

Proposed clodronate signaling pathway

Figure 1. Proposed clodronate signaling pathway

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