CYP7B1-mediated 25-hydroxycholesterol degradation maintains quiescence-activation balance and improves therapeutic potential of mesenchymal stem cells.

Stem cells remain quiescent in vivo and become activated in response to external stimuli. However, the mechanism regulating the quiescence-activation balance of bone-marrow-derived mesenchymal stem cells (BM-MSCs) is still unclear. Herein, we demonstrated that CYP7B1 was the common critical molecule that promoted activation and impeded quiescence of BM-MSCs under inflammatory stimulation. Mechanistically, CYP7B1 degrades 25-hydroxycholesterol (25-HC) into 7α,25-dihydroxycholesterol (7α,25-OHC), which alleviates the quiescence maintenance effect of 25-HC through Notch3 signaling pathway activation. CYP7B1 expression in BM-MSCs was regulated by NF-κB p65 under inflammatory conditions. BM-MSCs from CYP7B1 conditional knockout (CKO) mice had impaired activation abilities, relating to the delayed healing of bone defects. Intravenous infusion of BM-MSCs overexpressing CYP7B1 could improve the pathological scores of mice with collagen-induced arthritis. These results clarified the quiescence-activation regulatory mechanism of BM-MSCs through the NF-κB p65-CYP7B1-Notch3 axis and provided insight into enhancing BM-MSCs biological function as well as the subsequent therapeutic effect.

TNF-α-Induced KAT2A Impedes BMMSC Quiescence by Mediating Succinylation of the Mitophagy-Related Protein VCP.

Regular quiescence and activation are important for the function of bone marrow mesenchymal stem cells (BMMSC), multipotent stem cells that are widely used in the clinic due to their capabilities in tissue repair and inflammatory disease treatment. TNF-α is previously reported to regulate BMMSC functions, including multilineage differentiation and immunoregulation. The present study demonstrates that TNF-α impedes quiescence and promotes the activation of BMMSC in vitro and in vivo. Mechanistically, the TNF-α-induced expression of KAT2A promotes the succinylation of VCP at K658, which inhibits the interaction between VCP and MFN1 and thus inhibits mitophagy. Furthermore, activated BMMSC exhibits stronger fracture repair and immunoregulation functions in vivo. This study contributes to a better understanding of the mechanisms of BMMSC quiescence and activation and to improving the effectiveness of BMMSC in clinical applications.

Iron deficiency-induced ferritinophagy impairs skeletal muscle regeneration through RNF20-mediated H2Bub1 modification.

Iron deficiency (ID) is a widespread condition concomitant with disease and results in systemic dysfunction of target tissues including skeletal muscle. Activated by ID, ferritinophagy is a recently found type of selective autophagy, which plays an important role in various physiological and pathological conditions. In this study, we demonstrated that ID-mediated ferritinophagy impeded myogenic differentiation. Mechanistically, ferritinophagy induced RNF20 degradation through the autophagy-lysosomal pathway and then negatively regulated histone H2B monoubiquitination at lysine-120 in the promoters of the myogenic markers MyoD and MyoG, which inhibited myogenic differentiation and regeneration. Conditional knockout of NCOA4 in satellite cells, overexpression of RNF20 or treatment with 3-methyladenine restored skeletal muscle regenerative potential under ID conditions. In patients with ID, RNF20 and H2Bub1 protein expression is downregulated in skeletal muscle. In conclusion, our study indicated that the ferritinophagy-RNF20-H2Bub1 axis is a pathological molecular mechanism underlying ID-induced skeletal muscle impairment, suggesting potential therapeutic prospects.

Targeting macrophage M1 polarization suppression through PCAF inhibition alleviates autoimmune arthritis via synergistic NF-κB and H3K9Ac blockade.

Sustained inflammatory invasion leads to joint damage and progressive disability in several autoimmune rheumatic diseases. In recent decades, targeting M1 macrophage polarization has been suggested as a promising therapeutic strategy for autoimmune arthritis. P300/CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) that exhibits a strong positive relationship with the proinflammatory microenvironment. However, whether PCAF mediates M1 macrophage polarization remains poorly studied, and whether targeting PCAF can protect against autoimmune arthritis in vivo remains unclear. Commonly used drugs can cause serious side effects in patients because of their extensive and nonspecific distribution in the human body. One strategy for overcoming this challenge is to develop drug nanocarriers that target the drug to desirable regions and reduce the fraction of drug that reaches undesirable targets. In this study, we demonstrated that PCAF inhibition could effectively inhibit M1 polarization and alleviate arthritis in mice with collagen-induced arthritis (CIA) via synergistic NF-κB and H3K9Ac blockade. We further designed dextran sulfate (DS)-based nanoparticles (DSNPs) carrying garcinol (a PCAF inhibitor) to specifically target M1 macrophages in inflamed joints of the CIA mouse model via SR-A-SR-A ligand interactions. Compared to free garcinol, garcinol-loaded DSNPs selectively targeted M1 macrophages in inflamed joints and significantly improved therapeutic efficacy in vivo. In summary, our study indicates that targeted PCAF inhibition with nanoparticles might be a promising strategy for treating autoimmune arthritis via M1 macrophage polarization inhibition.

Super enhancers targeting ZBTB16 in osteogenesis protect against osteoporosis.

As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers (SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis development. Through integrated analysis, we identified the most common SE-targeted and osteoporosis-related osteogenic gene, ZBTB16. ZBTB16, positively regulated by SEs, promoted MSC osteogenesis but was expressed at lower levels in osteoporosis. Mechanistically, SEs recruited bromodomain containing 4 (BRD4) at the site of ZBTB16, which then bound to RNA polymerase II-associated protein 2 (RPAP2) that transported RNA polymerase II (POL II) into the nucleus. The subsequent synergistic regulation of POL II carboxyterminal domain (CTD) phosphorylation by BRD4 and RPAP2 initiated ZBTB16 transcriptional elongation, which facilitated MSC osteogenesis via the key osteogenic transcription factor SP7. Bone-targeting ZBTB16 overexpression had a therapeutic effect on the decreased bone density and remodeling capacity of Brd4fl/fl Prx1-cre mice and osteoporosis (OP) models. Therefore, our study shows that SEs orchestrate the osteogenesis of MSCs by targeting ZBTB16 expression, which provides an attractive focus and therapeutic target for osteoporosis. Without SEs located on osteogenic genes, BRD4 is not able to bind to osteogenic identity genes due to its closed structure before osteogenesis. During osteogenesis, histones on osteogenic identity genes are acetylated, and OB-gain SEs appear, enabling the binding of BRD4 to the osteogenic identity gene ZBTB16. RPAP2 transports RNA Pol II from the cytoplasm to the nucleus and guides Pol II to target ZBTB16 via recognition of the navigator BRD4 on SEs. After the binding of the RPAP2-Pol II complex to BRD4 on SEs, RPAP2 dephosphorylates Ser5 at the Pol II CTD to terminate the transcriptional pause, and BRD4 phosphorylates Ser2 at the Pol II CTD to initiate transcriptional elongation, which synergistically drives efficient transcription of ZBTB16, ensuring proper osteogenesis. Dysregulation of SE-mediated ZBTB16 expression leads to osteoporosis, and bone-targeting ZBTB16 overexpression is efficient in accelerating bone repair and treating osteoporosis.

Diet Control and Swimming Exercise Ameliorate HFD-Induced Cognitive Impairment Related to the SIRT1-NF-κB/PGC-1α Pathways in ApoE-/- Mice.

High-fat diet- (HFD-) induced neuroinflammation may ultimately lead to an increased risk of cognitive impairment. Here, we evaluate the effects of diet control and swimming or both on the prevention of cognitive impairment by enhancing SIRT1 activity. Twenty-week-old ApoE-/- mice were fed a HFD for 8 weeks and then were treated with diet control and/or swimming for 8 weeks. Cognitive function was assessed using the novel object recognition test (NORT) and Y-maze test. The expression of sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), brain-derived neurotrophic factor (BDNF), nuclear factor kappa B p65 (NF-κB p65), interleukin-1ß (IL-1ß), and tumour necrosis factor-α (TNF-α) in the hippocampus was measured by western blotting. The levels of fractional anisotropy (FA), N-acetylaspartate (NAA)/creatine (Cr) ratio, choline (Cho)/Cr ratio, and myo-inositol (MI)/Cr ratio in the hippocampus were evaluated by diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) using 7.0-T magnetic resonance imaging (MRI). Our results showed that cognitive dysfunction and hippocampal neuroinflammation appeared to be remarkably observed in apolipoprotein E (ApoE)-/- mice fed with HFD. Diet control plus swimming significantly reversed HFD-induced cognitive decline, reduced the time spent exploring the novel object, and ameliorated spontaneous alternation in the Y-maze test. Compared with the HFD group, ApoE-/- mice fed diet control and/or subjected to swimming had an increase in FA, NAA/Cr, and Cho/Cr; a drop in MI/Cr; elevated expression levels of SIRT1, PGC-1α, and BDNF; and inhibited production of proinflammatory cytokines, including NF-κB p65, IL-1ß, and TNF-α. SIRT1, an NAD+-dependent class III histone enzyme, deacetylases and regulates the activity of PGC-1α and NF-κB. These data indicated that diet control and/or swimming ameliorate cognitive deficits through the inhibitory effect of neuroinflammation via SIRT1-mediated pathways, strongly suggesting that swimming and/or diet control could be potentially effective nonpharmacological treatments for cognitive impairment.

The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting monocyte chemoattractant protein 1 (MCP1). However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, the N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regulated MCP1 expression in MSCs through the m6A modification. Specifically, the expression of METTL16 in MSCs decreased gradually and was negatively correlated with the expression of MCP1 after coculture with monocytes. Knocking down METTL16 markedly enhanced MCP1 expression and the ability to recruit monocytes. Mechanistically, knocking down METTL16 decreased MCP1 mRNA degradation, which was mediated by the m6A reader YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2). We further revealed that YTHDF2 specifically recognized m6A sites on MCP1 mRNA in the CDS region and thus negatively regulated MCP1 expression. Moreover, an in vivo assay showed that MSCs transfected with METTL16 siRNA showed greater ability to recruit monocytes. These findings reveal a potential mechanism by which the m6A methylase METTL16 regulates MCP1 expression through YTHDF2-mediated mRNA degradation and suggest a potential strategy to manipulate MCP1 expression in MSCs.

Decrotonylation of AKT1 promotes AKT1 phosphorylation and activation during myogenic differentiation.

INTRODUCTION: Myogenic differentiation plays an important role in pathophysiological processes including muscle injury and regeneration, as well as muscle atrophy. A novel type of posttranslational modification, crotonylation, has been reported to play a role in stem cell differentiation and disease. However, the role of crotonylation in myogenic differentiation has not been clarified. OBJECTIVES: This study aims to find the role of crotonylation during myogenic differentiation and explore whether it is a potential target in myogenic dysfunction disease. METHODS: C2C12 cell line and skeletal muscle mesenchymal progenitors of Mus musculus were used for myogenic process study in vitro, while muscle injury model of mice was used for in vivo muscle regeneration study. Mass spectrometry favored in discovery of potential target protein of crotonylation and its specific sites. RESULTS: We confirmed the gradual decrease in total protein crotonylation level during muscle differentiation and found decreased crotonylation of AKT1, which facilitated an increase in AKT1 phosphorylation. Then we verified that crotonylation of AKT1 at specific sites weakened its binding with PDK1 and impaired its phosphorylation. In addition, we found that increased expression of the crotonylation eraser HDAC3 decreased AKT1 crotonylation levels during myogenic differentiation, jointly promoting myogenic differentiation. CONCLUSION: Our study highlights the important role of decrotonylation of AKT1 in the process of muscle differentiation, where it aids the phosphorylation and activation of AKT1 and promotes myogenic differentiation. This is of great significance for exploring the pathophysiological process of muscle injury repair and sarcopenia.

LncRNA MRF drives the regulatory function on monocyte recruitment and polarization through HNRNPD-MCP1 axis in mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) exhibit two bidirectional immunomodulatory abilities: proinflammatory and anti-inflammatory regulatory effects. Long noncoding RNAs (lncRNAs) have important functions in the immune system. Previously, we performed high-throughput sequencing comparing lncRNA expression profiles between MSCs cocultured with or without CD14+ monocytes and screened out a new lncRNA termed lncRNA MCP1 regulatory factor (MRF). However, the mechanism of MRF in MSCs is still unknown. METHODS: MRF expression was quantified via qRT-PCR. RNA interference and lentiviruses were used to regulate MRF expression. The immunomodulatory effects of MSCs on monocytes were evaluated via monocyte migration and macrophage polarization assays. RNA pull-down and mass spectrometry were utilized to identify downstream factors of MRF. A dual-luciferase reporter assay was applied to analyze the transcription factors regulating MRF. qRT-PCR, western blotting and ELISAs were used to assess MCP1 expression. A human monocyte adoptive transfer mouse model was applied to verify the function of MRF in vivo. RESULTS: MRF was upregulated in MSCs during coculture with CD14+ monocytes. MRF increased monocyte recruitment by upregulating the expression of monocyte chemotactic protein (MCP1). Knockdown of MRF enhanced the regulatory effect of MSCs on restraining M1 polarization and facilitating M2 polarization. Mechanistically, MRF bound to the downstream protein heterogeneous nuclear ribonucleoprotein D (HNRNPD) to upregulate MCP1 expression, and the transcription factor interferon regulatory factor 1 (IRF1) activated MRF transcription early during coculture. The human monocyte adoptive transfer model showed that MRF downregulation in MSCs inhibited monocyte chemotaxis and enhanced the effects of MSCs to inhibit M1 macrophage polarization and promote M2 polarization in vivo. CONCLUSION: We identified the new lncRNA MRF, which exhibits proinflammatory characteristics. MRF regulates the ability of MSCs to accelerate monocyte recruitment and modulate macrophage polarization through the HNRNPD-MCP1 axis and initiates the proinflammatory regulatory process in MSCs, suggesting that MRF is a potential target to improve the clinical effect of MSC-based therapy or correct MSC-related immunomodulatory dysfunction under pathological conditions.

Osteogenic differentiation of mesenchymal stem cells promotes c-Jun-dependent secretion of interleukin 8 and mediates the migration and differentiation of CD4+ T cells.

BACKGROUND: The immune system and the skeletal system have complex interactions in the bone marrow and even in the joints, which has promoted the development of the concept of osteoimmunology. Some evidence has indicated that T cells and B cells contribute to the balance between the resorption and formation of bone. However, there has been little discussion on the regulation of CD4+ T lymphocytes by cells involved in bone metabolism. Mesenchymal stem cells (MSCs), which exert core functions related to immunoregulation and osteogenic differentiation, are crucial cells linked to both bone metabolism and the immune system. Previous studies have shown that the immunoregulatory capacity of MSCs changes following differentiation. However, it is still unclear whether the osteogenic differentiation of MSCs affects the migration and differentiation of CD4+ T cells. METHODS: MSCs were cultured in growth medium or osteogenic medium for 10 days and then cocultured with CD4+ T cells. CD4+ T cell migration and differentiation were detected by flow cytometry. Further, gene expression levels of specific cytokines were analyzed by quantitative real-time PCR and enzyme-linked immunosorbent assays. A Proteome Profiler Human XL Cytokine Array Kit was used to analyze supernatants collected from MSCs. Alizarin red S staining and Alkaline phosphatase assay were used to detect the osteogenic differentiation of MSCs. RESULTS: Here, we found that the migration of CD4+ T cells was elevated, and the capacity to induce the differentiation of regulatory T (Treg) cells was weakened during MSC osteogenic differentiation, while the differentiation of T helper 1 (Th1), T helper 2 (Th2) and T helper 17 (Th17) cells was not affected. Further studies revealed that interleukin (IL)-8 was significantly upregulated during MSC osteogenic differentiation. Both a neutralizing antibody and IL-8-specific siRNA significantly inhibited the migration of CD4+ T cells and promoted the differentiation of Treg cells. Finally, we found that the transcription factor c-Jun was involved in regulating the expression of IL-8 and affected the osteogenic differentiation of MSCs, thereby mediating the migration and differentiation of CD4+ T cells. CONCLUSION: This study demonstrated that MSC osteogenic differentiation promoted c-Jun-dependent secretion of IL-8 and mediated the migration and differentiation of CD4+ T cells. These results provide a further understanding of the crosstalk between bone and the immune system and reveal information about the relationship between osteogenesis and inflammation in the field of osteoimmunology.