Smad4-p65 interactions drive BMP-mediated protection against inflammatory cell death [ATAC-Seq]

Inflammation-induced osteoblast death undermines bone homeostasis and can aggravate bone-destructive diseases, yet how pro-survival BMP signaling intersects with inflammatory TNF–NF-?B signaling remains poorly defined. Here we show that BMP4 protects mouse osteoblasts from TNF-a–triggered apoptosis and cytotoxic death and that this cytoprotection requires both Smad4 and NF-?B p65. Integrative epigenomic profiling (ChIP-seq/ATAC-seq/RNA-seq) reveals extensive Smad4–p65 co-occupancy and identifies Samd9l as a prominent TNF-a–inducible gene that is selectively repressed by BMP4. Two Smad4/p65-bound distal elements physically contact the Samd9l promoter, exhibit TNF-responsive enhancer activity, and are required for Samd9l induction, as demonstrated by 4C-seq and CRISPR/dCas9-based chromatin closing/opening. Functionally, Samd9l depletion attenuates TNF-a–driven caspase activation and cytotoxicity in osteoblasts and enhances BMP2-driven ectopic bone formation in vivo. Cross-species mapping suggests that the two mouse regulatory elements converge into a single promoter-proximal composite element at the human SAMD9L locus, supporting a conserved inflammatory control node. Together, these findings define a BMP4–Smad4 mechanism that reshapes NF-?B enhancer outputs to limit osteoblast death and nominate the SAMD9L regulatory circuit as a therapeutic entry point for inflammatory bone loss. Overall design: Examination of the effects of TNFa and/or BMP4 on chromatin accessiblity in murine osteoblastic cells

炎症诱导的成骨细胞死亡会破坏骨稳态，并加剧骨破坏性疾病，但促存活的骨形态发生蛋白（Bone Morphogenetic Protein, BMP）信号通路与炎症性肿瘤坏死因子-核因子κB（TNF–NF-κB）信号通路的交互机制仍未得到充分阐明。本研究证实，骨形态发生蛋白4（BMP4）可保护小鼠成骨细胞免受肿瘤坏死因子α（TNF-α）触发的凋亡与细胞毒性死亡，且该细胞保护效应同时依赖于Smad4与NF-κB p65。整合表观基因组分析（染色质免疫共沉淀测序/转座酶可及性测序/RNA测序，ChIP-seq/ATAC-seq/RNA-seq）结果显示，Smad4与p65存在广泛的共占据现象，并鉴定出Samd9l作为受TNF-α诱导的核心基因，且该基因可被BMP4选择性抑制。两个被Smad4/p65结合的远端调控元件可与Samd9l启动子发生物理相互作用，表现出TNF响应性增强子活性，且是Samd9l诱导表达所必需的——这一结论通过染色体构象捕获测序（4C-seq）以及基于成簇规律间隔短回文重复序列/失活Cas9（CRISPR/dCas9）的染色质闭合/开放实验得到了验证。功能实验表明，敲低Samd9l可减弱成骨细胞中TNF-α介导的半胱天冬酶激活与细胞毒性，并在体内增强骨形态发生蛋白2（BMP2）诱导的异位成骨。跨物种映射分析显示，小鼠的两个调控元件可在人类SAMD9L基因座上汇聚为单个启动子近端复合调控元件，提示存在保守的炎症调控节点。综上，本研究阐明了一条BMP4–Smad4信号通路机制：该通路可重塑NF-κB增强子的调控输出活性，从而限制成骨细胞死亡，并将SAMD9L调控环路定为炎症性骨丢失的潜在治疗靶点。整体实验设计：检测TNF-α与/或BMP4对小鼠成骨细胞染色质可及性的影响。