Inhibition of Osteoblast Proliferation and Migration by Exogenous and Endogenous Formaldehyde

Exogenous and endogenous formaldehyde (FA) plays an important role in cell growth and migration; however, its potential role in osteoblasts remains largely unclear. Cell counting kit-8 (CCK-8) and wound healing assays revealed that FA exposure at naturally occurring concentrations inhibited the proliferation and migration of mouse preosteoblast MC3T3-E1 cells. Moreover, RNA sequencing (RNA-seq) analysis revealed that FoxO1 signaling pathway components displayed distinct expression patterns upon FA exposure, reflected through significant enrichment of cell migration. In particular, FoxO1 Sirt1 and FA-induced related protein expression which were closely with cell proliferation and migration were confirmed by western blotting. The present results indicate that the FoxO1 pathway is involved in FA-induced inhibition of cell growth and migration.


Introduction
Osteoblast growth and migration are essential for not only bone metabolism, including bone remodeling and responses to mechanical loading, but also bone pathological processes including bone repair after fractures and during osteoporosis [1][2][3].
Formaldehyde (FA) is one of the primary factors causing sick building syndrome. FA enters the body through inhalation from building materials, furniture, tobacco smoke, e-cigarettes, the sweetener aspartame, and, most directly, accidental consumption of methanol and insulation materials [4,5]. The WHO-recommended limit for indoor FA is 0.1 mg/m 3 . However, indoor FA concentrations often exceed this recommended threshold. The average concentration of occupational FA exposure in Chinese factories is 1.37 mg/m 3 , which also exceeds the recommended threshold [6]. Simultaneously, FA is present in the natural environment, thus making it difficult to prevent FA exposure [7]. Furthermore, FA is produced endogenously, being found ubiquitously in cells owing to enzymatic oxidative demethylation by-products.
Histones of the KDM1/JMJC or ABH enzyme family and the RNA and DNA demethylation machinery produce FA in the nucleus [8,9].
Moreover, FA can be endogenously produced by the action of neutrophil enzyme myeloperoxidase and N-demethylation, a common biochemical phenomenon. Endogenous FA is present in the blood at 50-100 μM [10][11][12].
Therefore, because of its abundance and chemical properties, formaldehyde can pose a significant risk to genomic integrity . However, limited information is available regarding the mechanisms underlying protection against FA at the cellular and organismal levels. It is unclear whether FA affects bone cells, especially osteoblasts and their proliferation and migration, which play essential roles in bone-associated diseases, e.g., bone repair during fracture and osteoporosis.
This study aimed to characterize the effect of formaldehyde on osteoblast growth and migration. First, we investigated the effect of FA on mouse preosteoblast MC3T3-E1 cells; thereafter, we attempted to determine the key signaling pathway affected in osteoblasts upon FA exposure via genome-wide transcriptional profiling analysis using RNAseq.

Cell culture
MC3T3-E1 cells were purchased from the ATCC (Manassas, VA, USA). Cells were cultured in a humidified atmosphere of 5% CO 2 and 95% air at 37℃ in an alpha modification of Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin [13].

Cell proliferation
Cell proliferation was assessed via a colorimetric tetrazolium saltbased assay, i.e., the cell counting-8 (CCK-8) assay. MC3T3-E1 cells were seeded at a density of 5000 cells/well into a 96-well plate and cultured in the absence or presence of FA at different concentrations (100μM -500 μM). Thereafter, the CCK-8 solution (Dojindo) was added to each well, and the plate was incubated at 37℃ in a CO 2 incubator for 3 h. The absorbance was then determined at 450 nm, using a microplate reader (Bio-Rad, Hercules, CA, USA).

Wound-healing assay
Cells were seeded in a 6-well plate the day before the assay, and cells were pre-treated with FA for 2 h, 6 h or 12 h before the wounds were inflicted on the cell monolayer. The wound was generated by scratching the cell monolayer with a pipette tip, and images were acquired at 0, 12, and 24 h after wounding. The degree of cell migration required to close the wound was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

RNA preparation
Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA, 15596026) on dry ice and processed in accordance with the manufacturer's instructions. To eliminate DNA, an aliquot of total RNA was treated with RQ1 RNase-Free DNase (Promega, WI, USA, M6101), followed by phenol/chloroform/isoamyl alcohol extraction and chloroform/isoamyl alcohol extraction, using Phase Lock Gel Light tubes (5 PRIME 2302800), followed by ethanol precipitation. Precipitated RNA was stored at -20℃ until use.

Illumina RNA-Seq library construction
Total RNA (20 mg) was used for poly(A)t selection using oligo(dT) magnetic beads (Invitrogen, CA, USA, 610-02), eluted in water, and used to generate an RNA-seq library, using the ScriptSeq kit (Epicentre, CA, USA SS10906). Libraries were amplified via polymerase chain reaction for 12-15 cycles and sequenced in two lanes on the HiSeq 2000 platform at BGI Genome Center (Shenzhen, China).

RNA sequencing
High-throughput sequencing was performed as paired-end 100 sequencing, using a HiSeq 2000 system (Illumina, San Diego, CA, USA).

Effect of FA on osteoblast proliferation
The effect of FA on proliferation in MC3T3-E1 osteoblasts was determined using the CCK-8 assay. As shown in Fig. 1 (Fig. 1).

Effect of FA on osteoblast migration
We investigated the effect of FA on the migration of MC3T3-E1 cells,

Identification of the candidate signaling pathway involved in cell proliferation and migration after FA treatment
To explore the potential mechanism underlying osteoblast proliferation and migration after FA treatment at the whole-genome level, These upregulated genes were found to be primarily involved in DNA damage repair, cell metabolism, and other signaling pathways, while the downregulated genes were primarily involved in p53, Hippo, autophagy, FoxO, Wnt, TGF-beta, and other signaling pathways (Fig. 3C).
Furthermore, analysis of the RNA-seq results via GSEA revealed that the FA-300 group significantly inhibited signaling pathways such as FoxO, p53, Wnt, and mTOR (p < 0.05, Dunnett multiple comparison test) (Fig.   3D). These results preliminarily indicate that the in vitro MC3T3-E1 osteoblast model of FA treatment is indeed reliable.

DISCUSSION
To our knowledge, this study is the first to report that FA, a product of cellular metabolism and a ubiquitous environmental toxin, suppressed osteoblastic cell proliferation and decreased cell migration at naturally occurring concentrations. Furthermore, a genome-wide transcriptional analysis via RNA-seq performed to explore the potential mechanism underlying FA stimulation revealed distinct expression patterns of components of the FoxO signaling pathway upon FA treatment, reflected through significantly decreased cell migration and proliferation.
Forkhead box O1 (FoxO1), also known as the forkhead rhabdomyosarcoma transcription factor (FKHR), belongs to the Forkhead box (FOX) family and is a key transcriptional regulator in cell proliferation, differentiation, and migration via a receptor tyrosine kinase signaling pathway [17,18]. Recent studies have reported that osteoblast growth and migration is regulated by FoxO factors [19,20]. FoxO1 is the primary regulator of redox balance and function in osteoblasts among three key members of the FoxO family, i.e., FoxO1, FoxO3a, and FoxO4.
Depletion of FoxO1 in osteoblasts reportedly resulted in decreased proliferation and bone volume in FoxO1 Ob -/mice [21,22]. In the present study, genome-wide expression profiling revealed that FOXO1 was

Conflicts of interest
There is no conflict of interests in this work.    MC3T3-E1 were seeded in 6-well plates and treated with formaldehyde (FA). FoxO1 expression levels and FA-induced related protein expression levels were determined via western blot analysis.