Coordinated activation and potential reciprocal regulation of TGF-β1/Smad and FAK/Src/ERK signaling in radiation-induced fibrotic transformation of human endometrial stromal cells

Background Radiation-induced endometrial injury can lead to stromal cell dysfunction and fibrotic remodeling; however, the molecular basis of this process has not been fully elucidated. This study investigated whether coordinated activation and potential reciprocal regulation of TGF-β1/Smad and FAK/Src/ERK signaling contributes to radiation-induced fibrotic activation of human endometrial stromal cells (HESCs). Methods HESCs were subjected to varying doses of X-ray irradiation. Cell viability, migration, fibrotic marker expression, myofibroblast-like transformation, and signaling pathway activation were evaluated using CCK-8 assay, wound-healing assay, RT-qPCR, Western blotting, immunofluorescence staining, and ELISA. Pharmacological inhibition was performed using SB431542, a TGF-β receptor inhibitor, and PF-573228, a FAK inhibitor, either alone or in combination. Results Increasing doses of X-ray irradiation impaired HESC viability while driving a fibrogenic expression profile characterized by elevated TGF-β1, α-SMA, COL1A1, Fibronectin, and CTGF levels. Radiation also enhanced cell migration and promoted an α-SMA-positive myofibroblast-like phenotype. Mechanistically, radiation activated both TGF-β1/Smad and FAK/Src/ERK signaling, as indicated by increased phosphorylation of Smad2/3, FAK, Src, and ERK. SB431542 suppressed Smad2/3 activation and partially reduced FAK/Src/ERK phosphorylation, whereas PF-573228 inhibited FAK/Src/ERK activation and partially attenuated Smad2/3 phosphorylation, suggesting possible reciprocal pathway regulation. Combined inhibition more effectively reduced radiation-induced HESC migration, fibrotic gene and protein expression, Fibronectin deposition, and secretion of TGF-β1 and Collagen I than either inhibitor alone. Conclusion These findings suggest that radiation-induced fibrotic activation of HESCs is associated with coordinated activation of TGF-β1/Smad and FAK/Src/ERK signaling. Pharmacological inhibition data support a possible reciprocal regulatory relationship between these two pathways. Further genetic and in vivo validation is required before dual-pathway modulation can be considered as a therapeutic strategy for radiation-associated endometrial fibrotic injury.