T-2 toxin inhibits osteoblastic differentiation and mineralization involving mutual regulation between Wnt signaling pathway and autophagy

Mycotoxins are most frequent contaminants in environment and agricultural production globally. The T-2 toxin of Fusarium species is the most toxic type of A trichothecene mycotoxins. T-2 toxin can accumulate in bone and cause bone development disorders. Osteoblast is the functional cell responsible for bone formation. Whereas, the mechanism of T-2 toxin toxicity on osteoblast remains unknown. In present study, MC3T3-E1 cells were used to investigate the effect of T-2 toxin on differentiation and mineralization and potential underlying mechanism. The results showed that T-2 toxin inhibited both osteoblastic differentiation and mineralization. T-2 toxin repressed Wnt signaling pathway both during osteoblastic differentiation and mineralization. T-2 toxin similarly promoted autophagy during osteoblastic differentiation, while inhibited it during osteoblastic mineralization. In addition, the activation of Wnt signaling pathway mitigated T-2 toxin-induced osteoblast impairment, while the inhibition of autophagy exacerbated it. Our results also indicated that there was a positive feedback loop between the Wnt signaling pathway and autophagy. This study can provide a better understanding for relieving T-2-related bone diseases.


Introduction
Mycotoxins are toxic metabolites produced by different kinds of fungi and widely exist in soil, water source and various agro-environmental matrices (Cao et al. 2022;Juraschek et al. 2022). T-2 toxin, a trichothecene mycotoxin produced by Fusarium species, is the most frequently-detected mycotoxin contaminant in cereals, livestock feed and other agricultural products worldwide. A study in the Czech Republic showed that the detection rate of T-2 toxin in spring barley samples was 88% (Pernica et al. 2022). In Croatia, 70% oats tested positive for sum T-2/HT-2 toxin (Kovac et al. 2022). According to a 6year survey of mycotoxins in animal feed, T-2 toxin was detected in 98.9% and 96.4% of complete feeds and total mixed ration respectively, with the highest content of up to 131 µg/kg (Twaruzek et al. 2021). T-2 toxin can be enriched in food chains due to the stable physicochemical property, eventually harm human health and agricultural development (Li et al. 2011). T-2 toxin could easily be absorbed by the intestine and mainly accumulates in skeletal system. T-2 toxin causes bone dysplasia and degenerative joint changes Yan et al. 2010) and is an environmental risk factor of many bone diseases represented by Kaschin-Beck disease. Nevertheless, the osteotoxic mechanism of T-2 toxin is not fully understood. The Wnt signaling pathway is mainly divided into two categories, namely the classic Wnt signaling pathway (Wnt/β-catenin signaling pathway) and the non-canonical Wnt signaling pathway. The classic Wnt signaling pathway (hereafter referred to as "Wnt signaling pathway") is a critical pathway in regulating bone formation. Wnt signaling pathway regulates osteoblastic differentiation and mineralization by bone formation factors osterix (OSX), runt-related transcription factor-2 (RUNX-2), collagen types I (Col-I) and osteocalcin (OCN). Our previous research indicated that T-2 toxin induced femur formation disorders with down-regulated Wnt signaling pathway in mice (Zhang et al. 2022). More
Then ALP staining and Alizarin red S staining were used for identi cation. Experimental grouping and processing are detailed in the Supplementary Table S1.

ALP staining
The cells were washed three times with PBS, then xed in 4% formaldehyde at room temperature for 30 minutes. After washing the cells three times with distilled water, add the BCIP/NBT working solutions according to the instructions (Beyotime, Jiangsu, China). The plate was incubated for 30 minutes at 37°C without light. ALP staining was observed and photographed by the light microscope with a photographic machine (Olympus, Tokyo, Japan).

Alizarin red S staining
The cells were washed three times with phosphate buffered saline (PBS), then xed in 95% ethanol at room temperature for 30 minutes. After washing the cells three times with distilled water, add the Alizarin Red S staining reagent (Solarbio, Beijing, China) for 20 minutes with gentle agitation. Alizarin red S staining was observed and photographed by the light microscope with a photographic machine (Olympus, Tokyo, Japan).

MDC staining
Monodansylcadaverine (MDC) staining was used to detect autophagic vacuoles. After washing the cells three times with distilled water, add the MDC working solutions according to the instructions (Beyotime, Jiangsu, China). The plate was incubated for 30 minutes at 37°C without light. After staining, the cells were washed three times with Assay Buffer. Cells were observed and photographed by the uorescent microscopy with a photographic machine (Niko, Tokyo, Japan).

Western blotting
The protein expressions were detected by western blotting. As described previously (Cao et al. 2019), the total cell protein was extracted according to instructions of protein extraction reagents kit (Biosharp, Hefei, China), and then protein concentration was determined with a BCA analysis kit (Biosharp, Hefei, China). Proteins were separated by SDS-PAGE and wet-transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk for 2 h, then incubated with primary antibody overnight at 4°C. Membranes were washed three times in PBST and incubated with appropriate secondary antibodies for 2 h at room temperature. The enhanced ECL reagent (Biosharp, Hefei, China) was used to detect the target protein. Amersham TM Imager 600 (GE Healthcare, Pittsburgh, USA) was used for quantitative analysis. Densitometry analysis of speci c bands was performed by Image J software. The antibody information is detailed in the Supplementary Table S2.

Data analysis
The data were presented as means ± SEM. One-way ANOVA was used followed by LSD (equal variances) or Dunnett's T3 (unequal variances) as a posttest to determine statistical signi cance. The data was plotted using Origin Pro 2021 (Origin, Northampton, USA). A p-value less than 0.05 was considered signi cant, while a value less than 0.01 was considered markedly signi cant.

T-2 toxin inhibited osteoblast cell vitality
As shown in Fig.1, T-2 toxin treatment changed the cell morphology and decreased density of MC3T3-E1 cell. T-2 toxin in concentrations from 4 to 80 nM signi cantly suppressed viability of MC3T3-E1 cell in a dose-dependent effect. Less than 2 nM T-2 toxin treatment had no signi cant effect on cell viability (P 0.05), 4nM T-2 toxin treatment signi cantly decreased cell viability (P < 0.05), 8nM T-2 toxin treatment extremely signi cantly decreased cell viability(P < 0.01). Thus, these concentrations were selected for use in the subsequent series of experiments.
3.2 T-2 toxin induced osteoblastic differentiation disorders with the inhibition of Wnt signaling pathway and the activation of autophagy As shown in Fig.2, the differentiation capacity of MC3T3-E1 cell was decreased after T-2 toxin treatment. The results obtained from ALP staining (Fig.2-A) and protein expressions of RUNX-2 and OSX ( Fig.2-C) corroborated with each other. As shown in Fig.2-D, T-2 toxin treatment decreased the expressions of Wnt and β-catenin and increased the expression of GSK-3β, indicating T-2 toxin repressed Wnt signaling pathway. Furthermore, MDC staining ( Fig.2-B) and detections of autophagy-related protein (Fig.2-E) showed that T-2 toxin treatment enhanced autophagy.

Wnt signaling pathway and autophagy together protect against T-2 toxin-induced osteoblastic differentiation disorders
To further determine the role of Wnt signaling pathway and autophagy in T-2 toxin-induced osteoblastic differentiation disorders, we conducted intervention experiments. Treatment with Wnt activator promoted autophagy (Fig.3-B and E) and alleviated T-2 toxin-induced osteoblastic differentiation disorders (Fig.3-A).
These results showed that Wnt signaling pathway and autophagy synergistically antagonized T-2 toxininduced differentiation disorders.

T-2 toxin induced osteoblastic mineralization disorders with the inhibition of Wnt signaling pathway and autophagy
As shown in Fig.4, the mineralization capacity of MC3T3-E1 cell was decreased after T-2 toxin treatment.
The results obtained from Alizarin red S staining (Fig.4-A) and protein expressions of Col-I and OCN ( Fig.4-C) corroborated with each other. As shown in Fig.4-D, T-2 toxin treatment decreased the expressions of Wnt and β-catenin and increased the expression of GSK-3β, indicating T-2 toxin repressed Wnt signaling pathway. Unlike in differentiation, MDC staining ( Fig.4-B) and detections of autophagy-related protein ( Fig.4-E) showed that T-2 toxin treatment suppressed autophagy during mineralization process.

Wnt signaling pathway and autophagy together protect against T-2 toxin-induced osteoblastic mineralization disorders
To further determine the role of Wnt signaling pathway and autophagy in T-2 toxin-induced osteoblastic mineralization disorders, we also conducted intervention experiments. Treatment with Wnt activator promoted autophagy (Fig.4-B and E) and alleviated T-2 toxin-induced osteoblastic mineralization disorders (Fig.4-A). 3-MA treatment blocked the osteogenic capacity of MC3T3-E1 cells, as indicated by Alizarin red S staining (Fig.4-A) and protein expressions of Col-I and OCN (Fig.4-C). Furthermore, 3-MA treatment further decreased the expressions of Wnt and β-catenin and increased the expression of GSK-3β ( Fig.4-D). These results showed that Wnt signaling pathway and autophagy synergistically antagonized T-2 toxin-induced mineralization disorders.

Discussion
Mycotoxin contamination of environment is a serious health risk that is receiving increasing attention throughout the world (Cao et al. 2020;Juraschek et al. 2022). T-2 toxin, the most toxic mycotoxin of A trichothecene mycotoxins, has attracted the most attention for the bone toxicity. However, the effect of T-2 toxin on osteoblast remains unclear. In this research, we investigated the effects of T-2 toxin on osteoblastic differentiation and mineralization, and the regulatory roles of Wnt signaling pathway and autophagy during this process. Results showed that T-2 toxin disturbed osteoblastic differentiation and mineralization. Meanwhile, T-2 toxin repressed Wnt signaling pathway during both differentiation and mineralization. T-2 toxin activated autophagy during differentiation and inhibited autophagy during mineralization. Intervention trials showed that up-regulated of Wnt signaling pathway mitigates T-2 toxininduced osteoblast damage while inhibition of autophagy aggravates it. Furthermore, the mutual regulation between Wnt signaling pathway and autophagy participated in the T-2 toxin-induced osteoblast impairment.
Bone formation and reparation are complex, involving differentiation and mineralization of osteoblast.
The dysfunction of osteoblast causes a variety of bone diseases such as osteoporosis 13 . MC3T3-E1 cell line is a murine preosteoblast cell line with the ability to differentiate and mineralize and has been extensively used to assess osteoblast function in vitro (Jie et al. 2018). Our study indicated that the cell viability of MC3T3-E1 cell was reduced in a dose-dependent manner after 4-80 nM T-2 toxin treatment. To further investigate the impairment of T-2 toxin on osteoblast, the effects of T-2 toxin on differentiation and mineralization were subsequently detected.
ALP staining was used to detect the differentiation of osteoblast after T-2 toxin treatment. RUNX-2 is the transcription factor required for osteoblastic differentiation. OSX is the osteogenic differentiation marker downstream of RUNX-2. RUNX-2 is essential in formation of precursor osteoblast from mesenchymal stem cell and OSX is critical for differentiation of RUNX-2-expressing pre-osteoblast into mature and functional osteoblast(Sinha and Zhou 2013). Unsurprisingly, T-2 toxin inhibits osteoblastic differentiation and expressions of differentiation markers with a dose-effect relation. We also found that T-2 toxin repressed Wnt signaling pathway and the activation of Wnt signaling pathway mitigated T-2 toxininduced differentiation impairment. When the Wnt signaling pathway is activated, β-catenin, binding to GSK-3β, is released and promoting osteoblastic differentiation. Activation of the Wnt signaling pathway has been used as a target for the treatment of many bone loss-related diseases (Onuora 2021). Though the detailed molecular mechanisms by which autophagy regulates osteoblastic differentiation remain unclear, several studies have shown that autophagy promotes osteoblastic differentiation (Li et al. 2018b;Liu et al. 2013). In present research, T-2 toxin activated autophagy and the inhibition of autophagy exacerbated osteoblast differentiation disorders. Evidently, autophagy is the protective role in T-2 toxininduced osteoblastic differentiation disorders.
Alizarin Red S staining was used as an indicator of mineralization to detect calcium deposition in cells. Col-I is a major component constituting the bone matrix and a marker of early mineralization. OCN is a non-collagenous protein synthesized by osteoblast maintaining normal mineralization (Dong et al. 2018). Our results demonstrated that T-2 toxin decreased the formation of mineralized nodules and reduced the expressions of mineralized markers. Moreover, T-2 toxin repressed Wnt signaling pathway and decreased autophagy. Same as differentiation period, the activation of Wnt signaling pathway mitigated T-2 toxininduced mineralization impairment, whereas the inhibition of autophagy exacerbated it. There has been little agreement about the effect of Wnt signaling pathway on mineralization. The Wnt signaling pathway promotes mineralization in MC3T3-E1 cell line, mouse primary osteoblast, and human osteoblast (Dong et al. 2020;Nash et al. 2015;Yun et al. 2015). Conversely, the opposite conclusions were reached in IDG-SW3 cell line and dental pulp cell, which may be related to different cell types and treatments (Li et al. 2018a;Zhou et al. 2019b). Studies have shown that autophagy de ciency reduces mineralization capacity, which is probably due to autophagy vacuoles could secrete apatite crystals in osteoblast as carriers (Nollet et al. 2014). In addition, low levels of autophagy also caused the accumulation of damaged molecules and organelles and subsequently leading to abnormal cellular function. Therefore, inhibition of autophagy exacerbated the disorders of T-2 toxin-induced osteoblastic mineralization.
There is somewhat controversial of the relationship between Wnt signaling pathway and autophagy. In a variety of cancer cells, Wnt signaling pathway and autophagy are mutually negatively regulated.
Autophagy represses Wnt signaling pathway by accelerating β-catenin degradation in colorectal carcinoma cells (Petherick et al. 2013). In turn, the inhibition of Wnt signaling pathway increased autophagic ux (Nager et al. 2018). Other studies, however, have reached different conclusions. A study of hepatic progenitor cells (HPCs) showed that inhibition of autophagy by downregulating of ATG 5 expression impaired differentiation of HPCs and inhibited activation of the Wnt signaling pathway, which was relieved by over-expression of β-catenin (Ma et al. 2019). It is suggested that autophagy promotes HPCs differentiation by activating Wnt signaling. Another study found that rapamycin (an autophagy activator) treatment up-regulated the Wnt signaling pathway in mouse aortic endothelial cell (Liu et al. 2021). While treatment with Wnt3a could not only active Wnt signaling pathway, but also enhance autophagy. Consistent with these studies, our results indicate that there is a positive feedback loop between the Wnt signaling pathway and autophagy.
Our current study also has several limitations. First, it is not clear why T-2 toxin promotes autophagy during osteoblastic differentiation stage but inhibits autophagy during mineralization stage. We speculate that autophagy may simply a protective mechanism during osteoblastic differentiation while participates in secretory function during osteoblastic mineralization. Second, although we found mutual regulation between Wnt signaling pathway and autophagy, we didn't explore speci c mechanisms, which will need further study.
In conclusion, T-2 toxin induced differentiation and mineralization impairment of osteoblast. Wnt signaling pathway and autophagy play a synergistic protective role in this process. This research provides new insights into the bone toxicity mechanism of T-2 toxin.