TGF-β receptor expression and Smad2 localization are cell density dependent in fibroblasts

PURPOSE. In nonconfluent cultures, TGF-β induces differentiation of corneal fibroblasts to myofibroblasts. However, in confluent cultures, few fibroblasts differentiate to myofibroblasts after TGF-β1 addition. This study investigated the hypothesis that functional TGF-β receptor expression is greater in low-density cultures and is decreased in confluent cultures. METHODS. Northern and western blot analyses were used to detect smooth muscle (SM) α-actin message and protein. ¹²⁵I-labeled TGF-β1 was used in a radioreceptor-binding assay as an index of functional receptors on the cell surface of rabbit corneal fibroblast cultures prepared either at high density (cell-cell contact) or low density (absence of contact). Cell lysates were analyzed by SDS-PAGE and autoradiography. Total TGF-β receptor expression was evaluated in western blot analysis. Smad2, a downstream effector of TGF- β receptor activation, was immunodetected. RESULTS. Low-density cultures expressed more SM α-actin mRNA and protein than high-density cultures, indicating that the low-density cells were differentiating into myofibroblasts. When ¹²⁵I-TGF-β1 was added to low- and high-density fibroblasts, fibroblasts plated at low density bound more than fibroblasts in high density, confluent cultures. Furthermore, after the cells differentiated into myofibroblasts, they continued to bind 125I-TGF-β1. Specificity of ¹²⁵I-TGF-β1 binding was demonstrated by complete inhibition by excess nonradioactive TGF-β1. Localization of Smad2 was correlated with SM α-actin induction: Smad was nuclear in low-density cells and cytoplasmic in high- density cells. After TGF-β1 treatment, Smad2 remained cytoplasmic in high- density cells but was localized to nuclei in cells that were nonconfluent. CONCLUSIONS. Low cell density is correlated with increased functional expression of TGF-β receptors and promotion of signal transmission from these receptors. Thus, conditions that decrease cell density such as wounding favor myofibroblast differentiation in response to TGF-β.