Inhibition of Osteoclastogenesis by LY90009 through Notch Signaling Modulation in Osteolytic Disease Models

Summary

Here, we present a protocol to evaluate the inhibitory effects of the γ-secretase inhibitor LY900009 on RANKL-induced osteoclastogenesis in bone marrow-derived macrophages. The protocol includes cytotoxicity assessment, functional assays, molecular analyses, and in vivo validation in a murine osteolytic model, with applications in the therapeutic development of osteolytic diseases.

Abstract

Dysregulated osteoclast activity profoundly contributes to the progression of osteolytic disease, yet the extent of therapeutic intervention modalities is constrained by adverse effects. This study investigated LY900009, a γ-secretase inhibitor, and its capacity to suppress RANKL-induced osteoclastogenesis through modulation of Notch signaling. Using bone marrow-derived macrophages, we performed comprehensive in vitro evaluations of cytotoxicity, differentiation, and functional assays. Gene and protein expression analyses using qPCR and western blotting were used to elucidate the underlying molecular mechanisms. The in vivo efficacy was corroborated using a murine lipopolysaccharide-induced bone resorption model. LY900009 inhibited RANKL-induced osteoclast formation and resorptive function in a dose-dependent manner without cytotoxicity at therapeutic concentrations. This treatment significantly reduced the number of TRAP-positive osteoclasts, impaired actin belt formation, and suppressed bone matrix degradation. The expression of osteoclast markers, including NFATc1, c-Fos, cathepsin K, and TRAP, was markedly downregulated. Mechanistically, LY900009 disrupted Notch signaling and the downstream MAPK/Akt pathways. In vivo administration substantially reduced LPS-induced bone destruction, improving bone parameters and reducing osteoclast presence. These findings demonstrate that LY900009 effectively inhibits osteoclastogenesis and bone resorption by modulating the Notch/MAPK/Akt signaling axis, making it a promising therapeutic candidate for osteolytic disorders.

Introduction

Bone homeostasis constitutes an exquisitely orchestrated physiological paradigm in vertebrates, harmonized by the delicate equilibrium between osteoblast-mediated bone formation and osteoclast-driven bone resorption¹. This intricate balance maintains skeletal integrity, biomechanical resilience, and metabolic fidelity throughout one's lifespan, dynamically responding to mechanical demands, endocrine perturbations, and pathological challenges². The bone remodeling cycle involves sequential phases of activation, resorption, transition, formation, and termination, each governed by intricate molecular cascades that ensur....

Protocol

All animal procedures were conducted in accordance with institutional guidelines and were approved by the Animal Care and Use Committee of the Central Hospital affiliated with Shandong First Medical University (approval number IACUC-2024-015). The study adhered to established ethical principles for animal research and ARRIVE guidelines, including animal housing in standard cages (5 mice/cage, 12-h light/dark cycle, ad libitum food/water) and randomization to groups

1. Bone marrow-derived macrophage isolation and culture

1. Euthanize 5-week-old male C57BL/6 mice (n=5 per isolation) by placing them in a CO

Results

The successful implementation of this protocol revealed that LY900009 exerted potent inhibitory effects on osteoclast formation and function through multiple interconnected mechanisms. Initial cytotoxicity assessment in bone marrow-derived macrophages demonstrated time-dependent increases in toxicity, with calculated IC₅₀ values of 4.44 µM, 3.66 µM, and 2.93 µM at 24 h, 48 h, and 96 h, respectively (Figure 1A,B). Importantly, these cytotoxic concentrations were su.......

Discussion

The present investigation provides compelling evidence that LY900009 represents a promising therapeutic agent for osteolytic diseases through its potent and selective inhibition of osteoclastogenesis, consistent with the growing interest in γ-secretase inhibitors for bone-related disorders^14,20,21. The findings demonstrate that this γ-secretase inhibitor effectively suppresses RANKL-induced osteoclast formation and bon.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well cell culture plates	Corning	3524	Immunofluorescence microscopy
6-well cell culture plates	Corning	3516	Cell culture and western blot
70% Ethanol	Sigma-Aldrich	E7023	Sterilization and disinfection
75% Ethanol	Sigma-Aldrich	E7148	RNA washing buffer
96-well cell culture plates	Corning	3596	Cell culture and assays
Aluminum filter (0.5 mm)	Bruker	N/A	Micro-CT scanning filter
Anti-CD11b-FITC antibody	BioLegend	101206	Flow cytometry - macrophage marker
Anti-cleaved Notch1 antibody (rabbit monoclonal)	Cell Signaling Technology	4147S	Western blot - Notch signaling
Anti-F4/80-PE antibody	BioLegend	123108	Flow cytometry - macrophage marker
Anti-fade mounting medium	Vector Laboratories	H-1000	Fluorescence microscopy mounting
Anti-Hes1 antibody (rabbit monoclonal)	Cell Signaling Technology	11988S	Western blot - Notch downstream target
Anti-NFATc1 antibody (mouse monoclonal)	Santa Cruz Biotechnology	sc-7294	Western blot - osteoclast master regulator
Anti-phospho-Akt (Ser473) antibody	Cell Signaling Technology	4060S	Western blot - PI3K/Akt signaling
Anti-phospho-ERK1/2 (Thr202/Tyr204) antibody	Cell Signaling Technology	4370S	Western blot - MAPK signaling
Anti-phospho-NF-κB p65 (Ser536) antibody	Cell Signaling Technology	3033S	Western blot - NF-κB signaling
Anti-phospho-p38 MAPK (Thr180/Tyr182) antibody	Cell Signaling Technology	4511S	Western blot - p38 MAPK signaling
Anti-β-actin antibody (mouse monoclonal)	Cell Signaling Technology	4970S	Western blot loading control
Automated imaging reader	Molecular Devices	ImageXpress Micro	Resorption pit quantification
BCA Protein Assay Kit	Pierce/Thermo Fisher	23225	Protein concentration determination
Bleach (10% sodium hypochlorite)	Sigma-Aldrich	425044	Cell removal from resorption plates
BSA (Bovine Serum Albumin)	Sigma-Aldrich	A7906	Blocking buffer for immunofluorescence
C57BL/6 mice (male, 5-8 weeks old)	Charles River Laboratories	27	In vitro BMM isolation and in vivo studies
Calcium oxalate test reagent	Sigma-Aldrich	C0350	Decalcification completion testing
CCK-8 reagent (Cell Counting Kit-8)	Dojindo Molecular Technologies	CK04	Cell viability assessment
Cell strainer (70 μm)	BD Falcon	352350	Bone marrow cell filtration
Centrifuge (refrigerated)	Eppendorf	5424R	Cell and sample preparation
Chloroform	Sigma-Aldrich	C2432	RNA extraction phase separation
CO2 chamber	Euthanex Corp	EX-075	Animal euthanasia
CTAn software	Bruker	Version 1.20	Micro-CT image analysis
DAPI (4',6-diamidino-2-phenylindole)	Sigma-Aldrich	D9542	Nuclear counterstaining
Dimethylformamide (DMF)	Sigma-Aldrich	D4551	TRAP staining solvent
DMSO (Dimethyl sulfoxide)	Sigma-Aldrich	D2650	Vehicle control preparation
ECL Western Blotting Substrate	Pierce/Thermo Fisher	32106	Protein detection in western blot
EDTA (10%, pH 7.4)	Sigma-Aldrich	E6758	Decalcification solution
Eosin Y	Sigma-Aldrich	E4009	H&E histological staining
Fast Red Violet LB salt	Sigma-Aldrich	F3381	TRAP staining chromogen
Fetal Bovine Serum (FBS)	Gibco/Thermo Fisher	16140-071	Cell culture supplement
Flow cytometer	BD Biosciences	FACSCalibur	BMM characterization
Fluorescence microscope	Olympus	IX73	Immunofluorescence imaging
G*Power software	Heinrich Heine University	Version 3.1	Statistical power analysis
Glass coverslips (22 × 22 mm)	Fisher Scientific	12-541-B	Immunofluorescence microscopy
GraphPad Prism	GraphPad Software	Version 9.0	IC50 calculation and graphing
Hematoxylin	Sigma-Aldrich	H3136	H&E histological staining
Hemocytometer	Hausser Scientific	3200	Cell counting
High-Capacity cDNA Reverse Transcription Kit	Applied Biosystems	4368814	cDNA synthesis from RNA
HRP-conjugated anti-mouse IgG	Cell Signaling Technology	7076S	Western blot secondary antibody
HRP-conjugated anti-rabbit IgG	Cell Signaling Technology	7074S	Western blot secondary antibody
Hydroxyapatite-coated 96-well plates	Corning BioCoat	354651	Bone resorption functional assay
ImageJ	NIH	Version 1.53e	Image analysis and quantification
Incubator (CO2)	Thermo Fisher	51033058	Cell culture maintenance
Infrared imaging system	Bio-Rad	ChemiDoc MP	Western blot detection
Inverted microscope	Olympus	CKX41	Cell culture monitoring, TRAP counting
Isopropanol	Sigma-Aldrich	I9516	RNA precipitation
LPS (Lipopolysaccharide from E. coli)	Sigma-Aldrich	L4391	In vivo inflammatory bone resorption
LY900009 (γ-secretase inhibitor)	Selleck Chemicals	S7999	Test compound - osteoclast inhibitor
M-CSF (Macrophage Colony-Stimulating Factor)	R&D Systems	416-ML-010	Macrophage survival and osteoclast differentiation
Micro-CT scanner	Bruker	SkyScan 1272	Bone microarchitecture analysis
Microplate reader	BioTek	Synergy H1	Absorbance measurements (CCK-8)
Microtome	Leica	RM2255	Histological sectioning
Naphthol AS-MX phosphate	Sigma-Aldrich	N5000	TRAP staining substrate
Needles (25G, 27G)	BD	305122, 305109	Bone marrow flushing, injections
NRecon software	Bruker	Version 1.7	Micro-CT image reconstruction
Paraffin wax	Sigma-Aldrich	P3683	Tissue embedding
Paraformaldehyde (4%)	Sigma-Aldrich	P6148	Cell and tissue fixation
PBS (Phosphate Buffered Saline)	Gibco/Thermo Fisher	14190-144	Cell washing and buffer preparation
Pentobarbital sodium	Sigma-Aldrich	P3761	Animal anesthesia and euthanasia
Phosphatase inhibitor cocktail	Roche	4906837001	Phosphorylation preservation
PowerUp SYBR Green Master Mix	Applied Biosystems	A25742	Quantitative real-time PCR
Protease inhibitor cocktail	Roche	11697498001	Protein degradation prevention
PVDF membrane (0.45 μm)	Bio-Rad	1620177	Western blot protein transfer
RANKL (Receptor Activator of NF-κB Ligand)	R&D Systems	462-TEC-010	Osteoclast differentiation induction
Real-time PCR system	Applied Biosystems	7500 Fast	Quantitative gene expression analysis
Rhodamine-phalloidin	Invitrogen/Thermo Fisher	R415	F-actin visualization
RIPA buffer	Cell Signaling Technology	9806S	Protein extraction
RNase-free water	Ambion/Thermo Fisher	AM9937	RNA dissolution and PCR
SDS-PAGE gels (10%)	Bio-Rad	4561036	Western blot protein separation
Skim milk powder	Bio-Rad	1706404	Western blot blocking
Sodium acetate buffer (0.1 M, pH 5.0)	Sigma-Aldrich	S2889	TRAP staining buffer
Sodium tartrate (50 mM)	Sigma-Aldrich	S4297	TRAP staining specificity
Spectrophotometer	Thermo Fisher	NanoDrop 2000	RNA/protein concentration measurement
SPSS	IBM Corporation	Version 28.0	Statistical analysis
Syringes (1 mL, 10 mL)	BD	309659, 309604	Bone marrow flushing, injections
T75 cell culture flasks	Corning	430641	Primary BMM culture
TBST (Tris-Buffered Saline with 0.1% Tween-20)	Bio-Rad	1706531	Western blot washing buffer
Transfer buffer	Bio-Rad	1704270	Western blot protein transfer
Triton X-100	Sigma-Aldrich	T8787	Cell permeabilization
TRIzol reagent	Invitrogen/Thermo Fisher	15596026	Total RNA extraction
Trypan blue (0.4%)	Sigma-Aldrich	T8154	Cell viability assessment
Trypsin-EDTA (0.25%)	Gibco/Thermo Fisher	25200-056	Cell harvesting and detachment
Western blot transfer system	Bio-Rad	Trans-Blot Turbo	Protein transfer to membrane
Xylene	Sigma-Aldrich	X3750	Tissue processing and clearing
α-MEM (Alpha Minimum Essential Medium)	Gibco/Thermo Fisher	12561-056	Cell culture medium