Unique expression and critical role of metallothionein 3 in the control of osteoclastogenesis and osteoporosis

Bone homeostasis is maintained by an intricate balance between osteoclasts and osteoblasts, which becomes disturbed in osteoporosis. Metallothioneins (MTs) are major contributors in cellular zinc regulation. However, the role of MTs in bone cell regulation has remained unexplored. Single-cell RNA sequencing analysis discovered that, unlike the expression of other MT members, the expression of MT3 was unique to osteoclasts among various macrophage populations and was highly upregulated during osteoclast differentiation. This unique MT3 upregulation was validated experimentally and supported by ATAC sequencing data analyses. Downregulation of MT3 by gene knockdown or knockout resulted in excessive osteoclastogenesis and exacerbated bone loss in ovariectomy-induced osteoporosis. Transcriptome sequencing of MT3 knockdown osteoclasts and gene set enrichment analysis indicated that the oxidative stress and redox pathways were enriched, which was verified by MT3-dependent regulation of reactive oxygen species (ROS). In addition, MT3 deficiency increased the transcriptional activity of SP1 in a manner dependent on intracellular zinc levels. This MT3-zinc-SP1 axis was crucial for the control of osteoclasts, as zinc chelation and SP1 knockdown abrogated the promotion of SP1 activity and osteoclastogenesis by MT3 deletion. Moreover, SP1 bound to the NFATc1 promoter, and overexpression of an inactive SP1 mutant negated the effects of MT3 deletion on NFATc1 and osteoclastogenesis. In conclusion, MT3 plays a pivotal role in controlling osteoclastogenesis and bone metabolism via dual axes involving ROS and SP1. The present study demonstrated that MT3 elevation is a potential therapeutic strategy for osteolytic bone disorders, and it established for the first time that MT3 is a crucial bone mass regulator.


Measurement of PINP and CTX-1
Blood was collected from mice and allowed to rest for 30 minutes before being centrifuged for 15 minutes at 4°C and 4000 rpm.The serum obtained was then used for analysis.Measurements were conducted using a PINP ELISA kit (AC-33F1, Immunodiagnostic Systems, USA) or a CTX-1 ELISA kit (AC-06F1, Immunodiagnostic Systems), following the manufacturer's instructions.A 4-parameter logistic curve fit was utilized to generate the mean calibration curve and calculate the concentrations of samples.
Calcein double labeling 8-week-old mice received intraperitoneal injections of 20 mg/kg calcein (Sigma-Aldrich), dissolved in 2% sodium bicarbonate, on the first and seventh days.Three days after the second injection, femurs were harvested and fixed in 4% paraformaldehyde, then embedded in methyl methacrylate (MMA).
MMA resin blocks were sliced and subjected to confocal microscopy to visualize calcein deposited bands.Bone formation rate (BFR), mineral apposition rate (MAR), and mineralized surface per bone surface (MS/BS) were measured as previously described 1 .

Bone resorption assay
BMMs were cultured on dentin slices (Immunodiagnostic Systems, Boldon, United Kingdom) in the presence of RANKL and M-CSF for nine days.Dentin slices were scanned using a Carl Zeiss LSM 5-PASCAL laser-scanning microscope, and the resultant images were analyzed using LSM 5 Image Browser software (Carl Zeiss Microimaging) for both the resorption area and pit depth.

Gene silencing and overexpression
For gene silencing, gene-specific siRNA oligonucleotides were procured from Invitrogen (Carlsbad, USA).BMMs were transfected with siRNA oligonucleotides (40 nM) using Lipofectamine RNAiMAX according to the provided protocol.Sp1 siRNA was purchased from Thermo Fisher Scientific (Assay ID: s74196).The sequences of siRNA oligonucleotides were presented in pairs.

Reverse-transcription quantitative real-time PCR
Real-time PCR was performed as previously described 1 .The PCR cycle was consisted of a denaturation phase at 95°C for 3 seconds and an amplification phase at 60°C for 33 seconds.mRNA levels of genes were calculated using the 2 −ΔΔCT method with normalization to Hprt levels.
Supplemental Table 1 provides primer sequences used for real-time PCR analyses.

Chromatin immunoprecipitation (ChIP)
ChIP was performed with the ChIP kit (Cell Signaling, #9005) following the protocol provided by the manufacturer.Briefly, cells were treated with 37% formaldehyde to crosslink proteins to DNA.
After incubation at room temperature for 10 minutes, glycine was added to stop the reaction.
Following nuclear extraction, micrococcal nuclease was added to digest DNA and sonication was performed for 15 cycles of 30 seconds to achieve DNA fragments ranging from 150 to 900 bp.DNA fragmentation was verified by electrophoresis.For the input control, 2% of the sonicated sample was reserved.For chromatin immunoprecipitation, sonicated samples were incubated with SP1 antibody or control IgG at 4°C overnight.After adding ChIP-grade protein G magnetic beads, samples were incubated 2 hours at 4°C with rotation.Precipitated beads were thoroughly washed and incubated in the ChIP elution buffer at 65°C for 45 minutes with gentle vortexing.Subsequently, 2 μl of Proteinase K was added, and the mixture was incubated at 65°C for 2 hours to digest proteins.The purified DNA was then subjected to DNA electrophoresis and qPCR.

Western blotting
Cell lysates were acquired by treating cells with RIPA lysis buffer.For the isolation of nuclear and cytoplasmic proteins, we used the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, USA), following the protocol furnished by the manufacturer.Equal amounts of proteins were subjected to western blotting as previously described 1 .

Confocal microscopy
Cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and subjected to a blocking step with 1% BSA in PBS.Subsequently, the cells were incubated at 4°C with the selected primary antibody.After rinsing with PBS, the cells were exposed to the secondary antibody for 2 hours, followed by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) to highlight nuclei.
The prepared slides were examined under a Zeiss LSM 700 laser-scanning microscope.