Phosphoproteome and Biological Evidence Revealed Abnormal Calcium Homeostasis in Keloid Fibroblasts and Induction of Aberrant Platelet Aggregation

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 Ca2+ 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.