Deregulation of AP-1 Proteins in Collagen Gel-induced Epithelial Cell Apoptosis Mediated by Low Substratum Rigidity

Abstract

In this study, we established that collagen gel, but not collagen gel coating, induced apoptosis exclusively in epithelial cell lines, which indicated that low substratum rigidity might trigger cell apoptosis. To confirm this, we used collagen gels with different rigidities due to cross-linking or physical disruption of collagen fibrils caused by sonication. We found that collagen gel-induced apoptosis was inversely correlated with substratum rigidity. Low substratum rigidity collagen gel-induced apoptosis was neither prevented by Bcl-2 overexpression nor preceded by mitochondrial release of cytochrome c. This suggested that the mitochondrial pathway was not involved in low substratum rigidity-induced apoptosis. Low substratum rigidity activated c-Jun N-terminal kinase (JNK) within 4 h, but it also rapidly down-regulated c-Jun within 1 h and triggered persistent aberrant expression of c-Fos for at least 24 h. Either reduced c-Jun expression or c-Fos overexpression induced apoptosis in several epithelial cells. Inhibiting low substratum rigidity-induced JNK activation prevented aberrant c-Fos expression but only partially blocked low substratum rigidity-induced apoptosis. Taking these results together, we conclude that low substratum rigidity collagen gel induced apoptosis in epithelial cells and that deregulated AP-1 proteins mediated that apoptosis, at least in part.

Mechanosensing machinery for cells under low substratum rigidity

“…Moreover, our recent study showed that low substratum rigidity-induced apoptosis is inversely correlated with substratum rigidity. Nevertheless, a significant difference of apoptosis ratio still exists when cells are cultured under substratum rigidity between 20 and 103 Pa (52), in which range ␤ 1 -integrin is neither activated nor clustered. We noted that other stretch-activated ion channels or capacitative calcium entries may be involved in detecting matrix rigidity at this range (7,8).…”

“…However, resent studies showed that sensing mechanical stimuli is also crucial for cells to detect several useful signals from the external environment during development and tumorigenesis (14,15,27,42,51). The three-dimensional collagen gel culture system has been used as a model to study ECM signaling because it provides not only biochemical signals but also biophysical properties (52,53). The elastic modulus of soft tissue is in the few hundreds to several thousands of pascals range that can be made by collagen gel (37,46,57).…”

“…Among numerous focal adhesion complex proteins, FAK is a key regulator (26). It transmits information from integrin to the various signaling pathways, including those that regulate cellular events such as cell shape, migration, growth, differentiation, and apoptosis (22,23,44,52). FAK is a 125-kDa cytoplasmic tyrosine kinase and is autophosphorylated at Tyr397 immediately after integrin clustering.…”

Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells’ osteogenic differentiation

“…35 D1 cells (1×10 6 per injection) were harvested and mixed with the collagen gel. Experimental groups included cells treated with 1 µM simvastatin (SIM), and 1 µM SIM mixed with Y27632 (10 µM), blebbistatin (Blebb) (10 µM), or cytochalasin D (CD) (0.5 µM), respectively, and untreated controls.…”

Three-dimensional culture models of mammary gland

“…57 For example, low substratum rigidity as been shown to trigger apoptosis in epithelial cells. 58 Artificial scaffold systems are ideal tools in which to study such principles in that they can be tailored to match the mechanical conditions encountered by tissues in vivo. This is an exciting area of research considering that the tumor microenvironment is known to be stiffer than normal tissue 59 and stem cells have been shown to direct their differentiation pathways depending on the nature of the mechanical environment.…”