MOE Key Laboratory of Tumor Molecular Biology and Institute of Life and Health Engineering, Jinan University, Guangzhou, Guangdong 510632, China.
The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong 510632, China.
J Proteome Res. 2021 May 7;20(5):2521-2532. doi: 10.1021/acs.jproteome.0c00984. Epub 2021 Mar 12.
Keloid is a benign tumor characterized by persistent inflammation, increased fibroblast proliferation, and abnormal deposition of collagen in the wound. The etiology of keloid is unclear. Here, we explored the phospho-signaling changes in human keloid fibroblasts via phosphoproteome mass spectrometry analysis. We found that comparative phosphoproteomics could statistically distinguish keloid from control fibroblasts. Differentially expressed phosphoproteins could predict the activation of known keloid-relevant upstream regulators including transforming growth factor-β1, interleukin (IL)-4, and IL-5. With multiple bioinformatics analyses, phosphorylated FLNA, TLN1, and VCL were significantly enriched in terms of calcium homeostasis and platelet aggregation. We biologically verified that keloid fibroblasts had a higher level of Ca influx than the control fibroblasts upon ionomycin stimulation. Via co-cultivation analysis, we found that human keloid fibroblasts could directly promote platelet aggregation. As suggested by PhosphoPath and gene set enrichment analysis, pFLNA was centered as the top phosphoproteins associated with keloid phenotypes. We validated that pFLNA was upregulated both in keloid fibroblasts and keloid tissue section, implicating its biomarker potential. In conclusion, we reported the first phosphoproteome on keloid fibroblasts, based on which we revealed that keloid fibroblasts had aberrant calcium homeostasis and could directly induce platelet aggregation.
瘢痕疙瘩是一种良性肿瘤，其特征为持续性炎症、成纤维细胞增殖增加以及胶原在伤口中的异常沉积。瘢痕疙瘩的病因尚不清楚。在这里，我们通过磷酸化蛋白质组质谱分析探讨了人瘢痕疙瘩成纤维细胞中的磷酸化信号变化。我们发现，比较磷酸化蛋白质组学可以从统计学上区分瘢痕疙瘩和成纤维细胞。差异表达的磷酸化蛋白可以预测已知与瘢痕疙瘩相关的上游调节剂（包括转化生长因子-β1、白细胞介素（IL）-4 和 IL-5）的激活。通过多种生物信息学分析，磷酸化 FLNA、TLN1 和 VCL 在钙稳态和血小板聚集方面显著富集。我们通过生物学验证发现，在离子霉素刺激下，瘢痕疙瘩成纤维细胞的 Ca 内流水平高于对照成纤维细胞。通过共培养分析，我们发现人瘢痕疙瘩成纤维细胞可以直接促进血小板聚集。根据 PhosphoPath 和基因集富集分析的结果，pFLNA 是与瘢痕疙瘩表型相关的最重要的磷酸化蛋白。我们验证了 pFLNA 在瘢痕疙瘩成纤维细胞和成纤维细胞组织切片中均上调，提示其具有作为生物标志物的潜力。总之，我们报告了第一个关于瘢痕疙瘩成纤维细胞的磷酸蛋白质组，根据该结果，我们揭示了瘢痕疙瘩成纤维细胞存在异常的钙稳态，并可以直接诱导血小板聚集。
