Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.
Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.
Avnet
Sofia
S
Laboratory for Pathophysiology, Istituti Ortopedici Rizzoli, v. di barbiano 1/10, 40136 Bologna, Italy. sofia.avnet@ior.it
Lamolinara
Annavera
A
Zini
Nicoletta
N
Solimando
Liliana
L
Quacquaruccio
Gianni
G
Granchi
Donatella
D
Maraldi
Nadir Mario
NM
Giunti
Armando
A
Baldini
Nicola
N
D000143:Acids / Q000378:metabolism; D016375:Antisense Elements (Genetics) / Q000493:pharmacokinetics; D016723:Bone Remodeling / Q000502:physiology*; D001862:Bone Resorption / Q000378:metabolism / Q000628:therapy*; D056657:Cathepsin K; D002403:Cathepsins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D010010:Osteoclasts / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism
D000143:Acids; D016375:Antisense Elements (Genetics); D012333:RNA, Messenger; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; C534132:Ctsk protein, mouse