TGF-beta receptor expression and smad2 localization are cell density dependent in fibroblasts

In nonconfluent cultures, TGF-beta induces differentiation of corneal fibroblasts to myofibroblasts. However, in confluent cultures, few fibroblasts differentiate to myofibroblasts after TGF-beta1 addition. This study investigated the hypothesis that functional TGF-beta receptor expression is greater in low-density cultures and is decreased in confluent cultures.

Northern and western blot analyses were used to detect smooth muscle (SM) a-actin message and protein. 125I-labeled TGF-beta1 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-beta receptor expression was evaluated in western blot analysis. Smad2, a downstream effector of TGF-beta receptor activation, was immunodetected.

Low-density cultures expressed more SM alpha-actin mRNA and protein than high-density cultures, indicating that the low-density cells were differentiating into myofibroblasts. When 125I-TGF-beta1 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-beta1. Specificity of 125I-TGF-beta1 binding was demonstrated by complete inhibition by excess nonradioactive TGF-beta1. Localization of Smad2 was correlated with SM alpha-actin induction: Smad was nuclear in low-density cells and cytoplasmic in high-density cells. After TGF-beta1 treatment, Smad2 remained cytoplasmic in high-density cells but was localized to nuclei in cells that were nonconfluent.

Low cell density is correlated with increased functional expression of TGF-beta 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-beta.