Diabetes mellitus (DM) and osteoporosis are two common diseases that may develop as a cause-and-effect relationship since the incidence of osteoporotic fractures is significantly increased in DM patients. However, the pathophysiology of diabetic osteoporosis is yet to be clearly understood. Iron overload has been reported to lead to bone loss and closely related to osteoporosis. In this study, we hypothesized that high glucose and high fat (HGHF) may induce osteoblastic ferroptosis for the pathogenesis of diabetic osteoporosis and explored the possible molecular mechanisms behind. Using the diabetic rat model established by HGHF feeding with a subsequent intraperitoneal injection of a single low dose of streptozocin, we found that the serum ferritin level (a biomarker for body iron store) was significantly elevated in HGHF-fed rats and the expression of SLC7A11 and GPX4 (inhibitory marker proteins for ferroptosis) was markedly attenuated in the bone tissue of the rats with diabetic bone loss as compared to the normal rats. In an osteoblast cell model, treatment of pre-osteoblastic MC3T3-E1 cells with high glucose and palmitic acid (HGPA) not only suppressed osteoblast differentiation and mineralization but also triggered ferroptosis-related osteoblastic cell death. m 6 A-seq revealed that m 6 A methylation on ASK1 was 80.9-fold higher in HGPA-treated cells. The expression of p-ASK1 and p-p38 was also significantly elevated in the HGPA-treated cells. Knockout of METTL3 (methyltransferaselike 3), one of the major m 6 A methyltransferases, in MC3T3-E1 cells not only abrogated HGPA-induced activation of ASK1-p38 signaling pathway but also attenuated the level of ferroptosis. Therefore, HGHF-induced ferroptosis in osteoblasts may be the main cause of osteoporosis in DM via activation of METTL3/ How to cite this article: Lin Y, Shen X, Ke Y, et al. Activation of osteoblast ferroptosis via the METTL3/ASK1-p38 signaling pathway in high glucose and high fat (HGHF)-induced diabetic bone loss.

HBx is a short-lived protein whose rapid turnover is mainly regulated by ubiquitin-dependent proteasomal degradation pathways. Our prior work identified BAF155 to be one of the HBx binding partners. Since BAF155 has been shown to stabilize other members of the SWI/SNF chromatin remodelling complex by attenuating their proteasomal degradation, we proposed that BAF155 might also contribute to stabilizing HBx protein in a proteasome-dependent manner. Here we report that BAF155 protected hepatitis B virus X protein (HBx) from ubiquitin-independent proteasomal degradation by competing with the 20S proteasome subunit PSMA7 to bind to HBx. BAF155 was found to directly interact with HBx via binding of its SANT domain to the HBx region between amino acid residues 81 and 120. Expression of either full-length BAF155 or SANT domain increased HBx protein levels whereas siRNA-mediated knockdown of endogenous BAF155 reduced HBx protein levels. Increased HBx stability and steady-state level by BAF155 were attributable to inhibition of ubiquitin-independent and PSMA7-mediated protein degradation. Consequently, overexpression of BAF155 enhanced the transcriptional transactivation function of HBx, activated protooncogene expression and inhibited hepatoma cell clonogenicity. These results suggest that BAF155 plays important roles in ubiquitin-independent degradation of HBx, which may be related to the pathogenesis and carcinogenesis of HBV-associated HCC.

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