IL-23 induces the expression of pro-osteogenic factors in osteoclasts

Abstract

Background The mechanism for the new bone formation in ankylosing spondylitis (AS) is still unclear. Although it has been demonstrated that IL-23 plays a pivotal role in the pathophysiology of AS, IL-23 has no direct effects on osteoblasts but modulates the function of osteoclasts.

Aims To explore whether IL-23 indirectly facilitates new bone formation through osteoclasts in AS, here we analyzed whether IL-23 enhances the expression levels of pro-osteogenic factors by osteoclasts.

Methods Mononuclear cells were harvested from mouse bone marrow and cultured in the presence of M-CSF (50 ng/ml) and RANKL (30 ng/ml) to trigger the production of osteoclasts. Protein and mRNA expression levels of Semaphorin 4D, Ephrin B2, BMP2, BMP6, SPHK1, HtrA1 and Wnt10b were measured using Western blot and qRT-PCR.

Results Primary mononuclear cells were transformed into osteoclasts with RANKL and M-CSF. The increased expression of NFATc1 and TRAP together with TRAP staining of>3 nuclei were used to identify mature osteoclasts. The mRNA expression levels of BMP2, Ephrin B2 and SPHK1 were enhanced by 1.46, 2.1 and 2.46 folds after exposure to IL-23. Confirmation of increased levels of Ephrin B2 and SPHK1 in IL-23-stimulated osteoclasts was provided by Western blot analysis. IL-23 had no effects on the expression of BMP6 or Wnt10b, or on the anti-osteogenic factors Semaphorin 4D or HtrA1.

Conclusions IL-23 induces osteoclasts to express pro-osteogenic factors rather than anti-osteogenic factors, suggesting IL-23 might indirectly promote the differentiation of osteoblasts through activated osteoclasts in ankylosing spondylitis.

Zusammenfassung

Hintergrund Der Mechanismus der Knochenneubildung bei ankylosierender Spondylitis (AS) ist nach wie vor unklar. Es wurde zwar nachgewiesen, dass IL-23 in der Pathophysiologie von AS eine zentrale Rolle spielt, doch IL-23 hat keine direkten Auswirkungen auf Osteoblasten, sondern moduliert die Funktion von Osteoklasten.

Ziele Es sollte untersucht werden, ob IL-23 bei AS indirekt eine Knochenneubildung durch Osteoklasten begünstigt. In diesem Zusammenhang wurde analysiert, ob IL-23 die Expressionsniveaus pro-osteogener Faktoren durch Osteoklasten steigert.

Methoden Es wurden mononukleäre Zellen aus dem Knochenmark von Mäusen gewonnen, die zur Auslösung der Produktion von Osteoklasten in Gegenwart von M-CSF (50 ng/ml) und RANKL (30 ng/ml) kultiviert wurden. Mithilfe von Western-Blots und qRT-PCR wurden die Protein- und mRNA-Expressionsniveaus von Semaphorin 4D, Ephrin B2, BMP2, BMP6, SPHK1, HtrA1 und Wnt10b gemessen.

Ergebnisse Primäre mononukleäre Zellen wurden mithilfe von RANKL und M-CSF in Osteoklasten umgewandelt. Die Identifikation reifer Osteoklasten erfolgte anhand der erhöhten Expression von NFATc1 und TRAP zusammen mit dem Nachweis von mehr als 3 Zellkernen in der TRAP-Färbung. Die mRNA-Expressionsniveaus von BMP2, Ephrin B2 und SPHK1 waren nach Exposition gegenüber IL-23 um das 1,46-, 2,1- und 2,46-Fache erhöht. Die Bestätigung der erhöhten Konzentration von Ephrin B2 und SPHK1 in IL-23-stimulierten Osteoklasten erfolgte durch Western-Blot-Analyse. IL-23 hatte keine Auswirkungen auf die Expression von BMP6 oder Wnt10b oder auf die anti-osteogenen Faktoren Semaphorin 4D oder HtrA1.

Schlussfolgerungen IL-23 induziert Osteoklasten eher zur Expression pro-osteogener als anti-osteogener Faktoren, was darauf schließen lässt, dass IL-23 bei ankylosierender Spondylitis durch aktivierte Osteoklasten die Differenzierung von Osteoblasten fördern könnte.

Key words

IL-23 - osteoclasts - ankylosing spondylitis - Ephrin B2 - sphingosine kinase 1

Schlüsselwörter

IL-23 - Osteoklasten - Spondylitis ankylosans - Ephrin B2 - Sphingosinkinase 1

Publikationsverlauf

Artikel online veröffentlicht:
11. Februar 2020

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