Neuroblastoma (NB) is the most common pediatric solid tumor that originates from neural crest-derived sympathoadrenal precursor cells that are committed to development of sympathetic nervous system. The well differentiated histological phenotype of NB tumor cells has been reportedly associated with favorable patient outcome. Retinoic acid (RA) can effectively induce NB cell differentiation, thereby being used in the clinic as a treatment agent for inducing the differentiation of high-risk NB. However, the underlying molecular mechanisms of regulating differentiation remain elusive. The correlation between clinical characteristics, survival and the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) expression were assessed using a neuroblastic tumor tissue microarray, and then validated in three independent patient datasets. The different expression of UCHL1 in ganglioneuroblastoma, ganglioneuroma and NB was detected by immunohistochemistry, mass spectra and immunoblotting analysis, and the correlation between UCHL1 expression and the differentiated histology was analyzed, which was also validated in three independent patient datasets. Furthermore, the roles of UCHL1 in NB cell differentiation and proliferation and the underlying mechanisms were studied by using short hairpin RNA and its inhibitor LDN57444 in vitro. Based on our neuroblastic tumor tissue microarrays and three independent validation datasets (Oberthuer, Versteeg and Seeger), we identified that UCHL1 served as a prognostic marker for better clinical outcome in NB. We further demonstrated that high UCHL1 expression was associated with NB differentiation, indicated by higher UCHL1 expression in ganglioneuroblastomas/ganglioneuromas and well-differentiated NB than poorly differentiated NB, and the positive correlation between UCHL1 and differentiation markers. As expected, inhibiting UCHL1 by knockdown or LDN57444 could significantly inhibit RA-induced neural differentiation of NB tumor cells, characterized by decreased neurite outgrowth and neural differentiation markers. This effect of UCHL1 was associated with positively regulating RA-induced AKT and ERK1/2 signaling activation. What’s more, knockdown of UCHL1 conferred resistance to RA-induced growth arrest. Our findings identify a pivotal role of UCHL1 in NB cell differentiation and as a prognostic marker for survival in patients with NB, potentially providing a novel therapeutic target for NB.

Excessive osteoclastogenesis is a key driver of inflammatory bone loss. Suppressing osteoclastogenesis has always been considered essential for the treatment of inflammatory bone loss. N-acetyltransferase 10 (NAT10) is the sole enzyme responsible for N4-acetylcytidine (ac4C) modification of mRNA, and is involved in cell development. However, its role in osteoclastogenesis and inflammatory bone loss remained elusive. We aimed to clarify the regulatory mechanism of NAT10 and ac4C modification in osteoclastogenesis and inflammatory bone loss. NAT10 expression and ac4C modification during osteoclastogenesis were determined by quantitative real-time PCR (qPCR), western blotting, dot blot and immunofluorescent staining, and the effect of NAT10 inhibition on osteoclast differentiation in vitro was measured by the tartrate-resistant acid phosphatase staining, podosome belts staining assay and bone resorption pit assay. Then, acRIP-qPCR and NAT10RIP-qPCR, ac4C site prediction, mRNA decay assay and luciferase reporter assay were performed to further study the underlying mechanisms. At last, mice models of inflammatory bone loss were applied to verify the therapeutic effect of NAT10 inhibition in vivo. NAT10 expression was upregulated during osteoclast differentiation and highly expressed in alveolar bone osteoclasts from periodontitis mice. Inhibition of NAT10 notably reduced osteoclast differentiation in vitro, as indicated by great reduction of tartrated resistant acid phosphatse positive multinuclear cells, osteoclast-specific gene expression, F-actin ring formation and bone resorption capacity. Mechanistically, NAT10 catalyzed ac4C modification of Fos (encoding AP-1 component c-Fos) mRNA and maintained its stabilization. Besides, NAT10 promoted MAPK signaling pathway and thereby activated AP-1 (c-Fos/c-Jun) transcription for osteoclastogenesis. Therapeutically, administration of Remodelin, the specific inhibitor of NAT10, remarkably impeded the ligature-induced alveolar bone loss and lipopolysaccharide-induced inflammatory calvarial osteolysis. Our study demonstrated that NAT10-mediated ac4C modification is an important epigenetic regulation of osteoclast differentiation and proposed a promising therapeutic target for inflammatory bone loss.

Background: Mechanosensitive ion channel PIEZOs have been widely reported to involve inflammation and pain. This study aimed to clarify expression patterns of PIEZOs and their potential relations to irreversible pulpitis. Materials and methods: Normal pulp tissues (n = 29) from patients with impacted third molars and inflamed pulp tissues (n = 23) from patients with irreversible pulpitis were collected. Pain levels were assessed using a numerical rating scale. PIEZO expressions were measured using real-time PCR and then confirmed using GEO datasets GSE77459, immunoblot, and immunohistochemistry staining. Correlations of PIEZO mRNA expression with inflammatory markers, pain markers, or clinical pain levels were evaluated using Spearman’s correlation analysis. Univariate analysis was conducted to analyze PIEZO expressions based on pain description and clinical examinations of cold test, percussion, palpation, and bite test. Results: Compared with normal pulp tissues, mRNA expression levels of PIEZO1 were significantly increased in inflamed pulp tissues, while PIEZO2 was significantly decreased, which was further confirmed in GSE77459 and on a protein and histological level. The positive correlation of the mRNA expression levels between PIEZO1 and inflammatory markers, as well as between PIEZO2 and pain markers, was verified. PIEZO2 expression was also positively correlated with pain levels. Besides, irreversible pulpitis patients who reported continuous pain and who detected a positive response to cold stimulus exhibited a higher expression level of PIEZO2 in the inflamed pulp tissues. By contrast, patients reporting pain duration of more than one week showed a higher expression level of PIEZO1. Conclusions: This study demonstrated the upregulation of PIEZO1 and the downregulation of PIEZO2 in irreversible pulpitis and revealed the potential relation of PIEZO1 and PIEZO2 to inflammation and pain. These findings suggested that PIEZOs might play critical roles in the progression of irreversible pulpitis and paved the way for further investigations aimed at novel therapies of irreversible pulpitis by targeting PIEZOs.

Aberrant angiogenesis is an important pathological feature of periodontitis, which is regulated by angiogenic paracrine factors derived from periodontal ligament stem cells (PDLSCs). We have previously demonstrated that ubiquitin C-terminal hydrolase L1 (UCHL1) was upregulated in PDLSCs from periodontitis patients, but its role in aberrant angiogenesis in periodontitis remains unclear. PDLSCs were isolated from healthy individuals and periodontitis patients. To mimic inflammatory conditions in vitro, PDLSCs from healthy individuals were treated with tumor necrosis factor-alpha and interleukin-1beta. Human umbilical vein endothelial cells were cultured with conditioned media from PDLSCs, and their proliferation, migration, and tube formation were detected to evaluate the pro-angiogenic capacity of PDLSCs. Vascular endothelial growth factor A (VEGFA) and angiopoietin 1 (ANGPT1) levels were measured using RT-qPCR, Western blotting, Immunofluorescence, and enzyme-linked immunosorbent assay. UCHL1 was knocked down using shRNA to validate its function and regulation of Yes-associated protein (YAP) activity. Co-immunoprecipitation was used to verify the interaction of UCHL1 with hypoxia-inducible factor 1 alpha (HIF-1α), and chromatin immunoprecipitation was used to analyze the effect of HIF-1α on YAP transcription. UCHL1 inhibitor was administered in a periodontitis murine model to investigate its function in vivo. The pro-angiogenic capacity of PDLSCs from periodontitis patients was enhanced, accompanied by increased expressions of VEGFA and ANGPT1, which were positively correlated with UCHL1 expression. UCHL1 knockdown in PDLSCs abrogated the increased secretion of VEGFA and ANGPT1 and the enhanced pro-angiogenic capacity under inflammatory conditions. Mechanistically, UCHL1 functioned through facilitating YAP expression and nuclear translocation, a process that was mediated by the stabilization and activation of HIF-1α, which in turn promoted YAP transcription. In vivo inhibition of UCHL1 in a periodontitis murine model alleviated aberrant angiogenesis and reduced the expressions of VEGFA and ANGPT1, thus attenuating periodontitis progression by reducing lymphocyte infiltration, inflammatory cytokine levels, and alveolar bone resorption. This study demonstrated that UCHL1 promoted the pro-angiogenic capacity of PDLSCs in periodontitis through the HIF-1α/YAP signaling, which provides insights into the pathogenesis of periodontitis and paves the way for novel treatment strategies.

LL-37, an antimicrobial peptide with dual pro-/anti-inflammatory roles, has been implicated in the pathogenesis of periodontitis, yet its specific action through its receptor Formyl peptide receptor 2 (FPR2) remains unclear. This study aimed to elucidate the expression profile of the LL-37/FPR2 axis in periodontitis and its regulatory effects on gingival fibroblasts (GFs). Fifty-seven periodontitis patients and fifty-seven healthy controls were included to assess the levels of LL-37 and FPR2 in gingival tissues, gingival crevicular fluid and GFs, as well as to assess their correlation with disease progression. Primary human GFs were isolated and subsequently stimulated with Porphyromonas gingivalis lipopolysaccharide alongside varying concentrations of LL-37. To elucidate the functional role of FPR2 in this system, targeted gene silencing was performed using small interfering RNA. Both LL-37 and FPR2 were upregulated in gingival tissues or gingival crevicular fluid from periodontitis patients, showing a significant positive correlation. FPR2 expression increased in GFs under periodontal inflammatory conditions, and high concentration of LL-37 exacerbated pro-inflammatory response in GFs via FPR2. Clinically, elevated LL-37 and FPR2 correlated with disease severity, particularly in patients with higher LL-37 expression. Patients with co-upregulation of both LL-37 and FPR2 exhibited more severe periodontitis. The LL-37/FPR2 axis was co-upregulated in periodontitis-affected gingival tissues and correlated with disease severity. The LL-37/FPR2 axis exacerbated pro-inflammatory response in GFs under periodontal inflammatory conditions, suggesting its potential as a therapeutic target for mitigating periodontal inflammation. We found that the co-upregulation of LL-37/FPR2 in periodontitis-affected gingival tissues, positively correlates with clinical parameters (PD/CAL). These findings provide new insights into the dual role of LL-37 in periodontal inflammation and suggest novel strategies for targeted therapy by selectively inhibiting high-concentration LL-37/FPR2 signaling.