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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Petridou
S
S
Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
Maltseva
O
O
Spanakis
S
S
Masur
S K
SK
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002452:Cell Count; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D051899:Smad2 Protein; D015534:Trans-Activators / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
D000199:Actins; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta