**Background:** Posterior capsule opacification (PCO) is the most common long-term complication of cataract surgery, caused by residual lens epithelial cells undergoing epithelial-to-myofibroblast transition (EMyT) driven by TGFβ. While TGFβ is known to require ERK signaling for EMyT, the upstream mechanisms linking TGFβ to ERK activation in lens cells were unclear. ErbB receptor tyrosine kinases had been implicated, but their role in TGFβ-induced ERK activation and EMyT had not been mechanistically defined.
**Methods:** The study used serum-free primary cultures of embryonic chick lens epithelial cells (DCDMLs), which mimic the avascular lens environment. Cells were treated with TGFβ1 (4 ng/ml) with or without various kinase inhibitors (lapatinib, erlotinib, afatinib for ErbB; UO126 for MEK/ERK; SB-431542 for TGFβR; PD173074 for FGFR; crenolanib for PDGFR; noggin for BMPR; sorafenib for Raf; SB203580 for p38) and antioxidants (NAC, GEE). Protein expression was analyzed by Western blot and immunofluorescence. Cell surface ErbB activation was assessed by biotinylation and phosphotyrosine immunoblotting. Ligand shedding was tested using conditioned medium from proHB-EGF-transfected DCDMLs on HEK293 cells. Statistical significance was determined using two-tailed paired Student's t-test.
**Key Results:** (1) Both ErbB inhibitors (lapatinib, erlotinib, afatinib) and the MEK inhibitor UO126 blocked TGFβ-induced expression of fibronectin (FN) and αSMA, with lapatinib achieving near-complete inhibition (p=0.000, n≥6). (2) ErbB inhibitors did not block TGFβ-induced Smad3 phosphorylation or lens fiber cell differentiation (δ-crystallin, CP115, CP49). (3) Lapatinib reduced basal pERK levels by >80% within 90 min, more than FGFR inhibition alone; combining lapatinib with FGFR inhibitor reduced pERK to levels comparable to UO126 (p≤0.001). (4) TGFβ increased pERK ~2-fold within 1.5 h, and this was blocked by lapatinib, erlotinib, or UO126, but not by PDGFR or BMPR inhibitors. FGFR inhibition partially reduced (~30%) TGFβ-induced pERK. (5) TGFβ did not increase ErbB autophosphorylation at 5, 45, or 90 min as assessed by cell surface biotinylation or pY1068-ErbB1 immunoblotting (p>0.2, n=3). (6) TGFβ did not stimulate HB-EGF shedding from DCDMLs. (7) The antioxidant NAC (20 mM) blocked TGFβ-induced pERK elevation (but not FGF- or HB-EGF-induced pERK) and prevented TGFβ-induced FN and αSMA expression after 6 days, without affecting δ-crystallin. (8) Prolonged TGFβ exposure (6 days) increased both pERK and total ERK levels ~2-fold, and sensitized cells to H2O2-induced pERK (8.56-fold increase vs. no TGFβ, p=0.000). (9) ERK inhibition with UO126 reduced basal and TGFβ-induced ErbB1 and ErbB2 protein levels (p=0.000), but not ErbB4; p38 inhibition increased ErbB1. (10) The Raf inhibitor sorafenib, MEK inhibitor UO126, and ErbB inhibitor lapatinib all blocked TGFβ-induced pERK, but not sodium orthovanadate-induced pERK, indicating TGFβ acts upstream of Raf in the canonical ErbB-Raf-MEK-ERK cascade.
**Clinical Implications:** This study identifies a novel TGFβ/ErbB/ERK signaling axis required for lens epithelial cell myofibroblast transition, the cellular basis of fibrotic PCO. The finding that ErbBs are the dominant source of basal ERK activity in lens cells suggests that ErbB inhibitors (e.g., lapatinib) could be as effective as global ERK inhibitors in preventing PCO initiation, with potentially fewer off-target effects in other ocular tissues where ERK is driven by non-ErbB pathways. The requirement for oxidative stress in TGFβ-induced ERK activation also suggests antioxidant strategies might mitigate PCO. Given the widespread role of TGFβ, oxidative stress, ERK, and myofibroblasts in fibrosis of lung, kidney, and liver, this TGFβ/ErbB/ERK axis may be relevant to fibrotic diseases beyond the lens.