Regulatory Role of SLIT3 in Osteoclast Differentiation

다국어 초록 (Multilingual Abstract)

Toll-like receptor 4 (TLR4) activation limits osteoclastogenesis directly or indirectly and thus affects bone homeostasis associated with infection and inflammation. The direct stimulation of TLR4 on osteoclast precursors (OCPs) inhibits RANKL-mediated osteoclastogenesis, but little is known about the underlying mechanisms.
Here, we show that TLR4 activation by lipopolysaccharide (LPS) induces concomitant expression of miR-218-2-3p and its host gene, SLIT3, in mouse bone marrow-derived macrophages (BMMs), which are OCPs. Knockdown of SLIT3 by RNA interference enhanced RANKL-mediated osteoclast (OC) differentiation after LPS stimulation, as determined by the OC marker genes TRAP, NFATc1, C/EBP1α, Tnfrsrf11a (RANK), and Cathepsin K (Ctsk). SLIT3 knockdown led to decreased miR-218-2-3p expression and increased Tnfrsf11a (RANK) expression in LPS-stimulated BMMs. Blocking endogenous miR-218-2-3p using an anti-miR also rescued the expression of RANK and subsequent OC formation in LPS-stimulated BMMs.
Thus, our findings demonstrate that TLR4-dependent induction of SLIT3 with concomitant expression of miR-218-2-3p targets RANK gene transcripts in OCPs and thereby restrains OC formation. These observations provide a mechanistic explanation for the regulatory role of TLR4 in the OC differentiation program.

목차 (Table of Contents)

• Introduction 1
• Materials and Methods 4
• Cell culture 4
• Reverse transcription (RT)-PCR analysis and Real time-PCR (qPCR) 4
• Western Blotting 5
• Gene silencing and transfection 5
• Immunofluorescence Confocal Microscopy 5
• Enzyme-linked immunosorbent assay (ELISA) for SLIT3 6
• Fluorescence-activated cell sorting (FACS) analysis 6
• Cloning and expression of SLIT3 LRR2 domain in E.coli 7
• Osteoclast differentiation 7
• Plasmid constructs 7
• Luciferase reporter assay 8
• Statistical analysis 8
• Results 9
• Enhancement of SLIT3 gene expression with concomitant expression of miR218-2-3p is dependent on TLR4 activation 9
• SLIT3 signal axis limits LPS-induced osteoclastogenesis 10
• Depletion of SLIT3 signal axis increases the expression of OC differentiation regulator and marker genes 11
• SLIT3 restrains the expression of RANK in LPS-stimulated primary macrophages 11
• Concomitant miR218-2-3p expression regulates RANK directly by targeting its 3’-UTR 12
• Discussion 13
• 국문요약 16
• References 26