Nuclear DNA, which is essential for the transmission of genetic information, is constantly damaged by external stresses and is subsequently repaired by the removal of the damaged region, followed by resynthesis of the...Nuclear DNA, which is essential for the transmission of genetic information, is constantly damaged by external stresses and is subsequently repaired by the removal of the damaged region, followed by resynthesis of the excised region. Accumulation of DNA damage with failure of repair processes leads to fatal diseases such as cancer. Recent studies have suggested that intra- and extra-nuclear environments play essential roles in DNA damage. However, numerous questions regarding the role of the nuclear mechanical environment in DNA damage remain unanswered. In this study, we investigated the effects of cell confluency (cell crowding) on the morphology of cell nuclei, and cytoskeletal structures, and DNA damage in NIH3T3 skin fibroblasts and HeLa cervical cancer cells. Although nuclear downsizing was observed in both NIH3T3 and HeLa cells with cell crowding, intracellular mechanical changes in the two cell types displayed opposite tendencies. Cell crowding in NIH3T3 cells induced reinforcement of actin filament structures, cell stiffening, and nuclear downsizing, resulting in a significant decrease in endogenous DNA damage, whereas cell crowding in HeLa cells caused partial depolymerization of actin filaments and cell softening, inducing endogenous DNA damage. Ultraviolet (UV) radiation significantly increased DNA damage in NIH3T3;however, this response did not change with cell crowding. In contrast, UV radiation did not cause DNA damage in HeLa cells under either sparse or confluent conditions. These results suggested that cell crowding significantly influenced endogenous DNA damage in cells and was quite different in NIH3T3 and HeLa cells. However, cell crowding did not affect the UV-induced DNA damage in either cell type.展开更多
Vascular smooth muscle cells (VSMCs) in the arterial walls play important roles in regulating vascular contraction and dilation. VSMCs actively remodel the arterial walls and dedifferentiate from the contractile to th...Vascular smooth muscle cells (VSMCs) in the arterial walls play important roles in regulating vascular contraction and dilation. VSMCs actively remodel the arterial walls and dedifferentiate from the contractile to the synthetic phenotype under pathological conditions. The mechanism underlying phenotypic transition of VSMCs is important for understanding its role in the pathophysiology of disease. Although numerous studies have reported various biochemical pathways that stimulate the phenotypic transition of VSMCs, very little is known about relation between their phenotypic transition and cellular traction force, which affects many cellular functions. In this study, we induced the differentiation of cultured VSMCs from the synthetic to the contractile phenotype by a low-serum cultivation and investigated changes in the cell traction forces using traction force microscopy technique. The expression of α-SMA, a contractile phenotype marker protein, was significantly upregulated with maturation of actin stress fibers in the low-serum culture, indicating VSMC differentiation was promoted in our experiments. The cells changed their morphology to an elongated bipolar shape, and the direction of the cell traction forces tended to align in the direction of the cell’s major axis. Despite the promotion of contractile differentiation in VSMCs, the overall cell traction forces were significantly reduced, indicating that excessive cell mechanical tension, which might induce cell proliferation and migration, was suppressed during contractile differentiation. These results suggest that suppression of cell traction force and enhanced force polarity might be key factors in VSMC differentiation induced by low serum culture.展开更多
Cells sense the external environment such as a surface topography and change many cellular functions. Cell nucleus has been proposed to act as a cellular mechanosensor, and the changes in nuclear shape possibly affect...Cells sense the external environment such as a surface topography and change many cellular functions. Cell nucleus has been proposed to act as a cellular mechanosensor, and the changes in nuclear shape possibly affect the functional regulation of cells. This study demonstrated a large-scale mechanical deformation of the intracellular nucleus using polydimethylsiloxane (PDMS)-based micropillar substrates and investigated the effects of nuclear deformation on migration, proliferation, and differentiation of vascular smooth muscle cells (VSMCs). VSMCs spread completely between the fibronectin-coated pillars, leading to strong deformations of their nuclei resulted in a significant inhibition of the cell migration. The proliferation and smooth muscle differentiation of VSMCs with deformed nuclei were dramatically inhibited on the micropillars. These results indicate that the inhibition of proliferation and VSMC differentiation resulted from deformation of the nucleus with high internal stress, and this type of large-scale nuclear mechanical stress might lead the cells to a “quiescent state”.展开更多
文摘Nuclear DNA, which is essential for the transmission of genetic information, is constantly damaged by external stresses and is subsequently repaired by the removal of the damaged region, followed by resynthesis of the excised region. Accumulation of DNA damage with failure of repair processes leads to fatal diseases such as cancer. Recent studies have suggested that intra- and extra-nuclear environments play essential roles in DNA damage. However, numerous questions regarding the role of the nuclear mechanical environment in DNA damage remain unanswered. In this study, we investigated the effects of cell confluency (cell crowding) on the morphology of cell nuclei, and cytoskeletal structures, and DNA damage in NIH3T3 skin fibroblasts and HeLa cervical cancer cells. Although nuclear downsizing was observed in both NIH3T3 and HeLa cells with cell crowding, intracellular mechanical changes in the two cell types displayed opposite tendencies. Cell crowding in NIH3T3 cells induced reinforcement of actin filament structures, cell stiffening, and nuclear downsizing, resulting in a significant decrease in endogenous DNA damage, whereas cell crowding in HeLa cells caused partial depolymerization of actin filaments and cell softening, inducing endogenous DNA damage. Ultraviolet (UV) radiation significantly increased DNA damage in NIH3T3;however, this response did not change with cell crowding. In contrast, UV radiation did not cause DNA damage in HeLa cells under either sparse or confluent conditions. These results suggested that cell crowding significantly influenced endogenous DNA damage in cells and was quite different in NIH3T3 and HeLa cells. However, cell crowding did not affect the UV-induced DNA damage in either cell type.
文摘Vascular smooth muscle cells (VSMCs) in the arterial walls play important roles in regulating vascular contraction and dilation. VSMCs actively remodel the arterial walls and dedifferentiate from the contractile to the synthetic phenotype under pathological conditions. The mechanism underlying phenotypic transition of VSMCs is important for understanding its role in the pathophysiology of disease. Although numerous studies have reported various biochemical pathways that stimulate the phenotypic transition of VSMCs, very little is known about relation between their phenotypic transition and cellular traction force, which affects many cellular functions. In this study, we induced the differentiation of cultured VSMCs from the synthetic to the contractile phenotype by a low-serum cultivation and investigated changes in the cell traction forces using traction force microscopy technique. The expression of α-SMA, a contractile phenotype marker protein, was significantly upregulated with maturation of actin stress fibers in the low-serum culture, indicating VSMC differentiation was promoted in our experiments. The cells changed their morphology to an elongated bipolar shape, and the direction of the cell traction forces tended to align in the direction of the cell’s major axis. Despite the promotion of contractile differentiation in VSMCs, the overall cell traction forces were significantly reduced, indicating that excessive cell mechanical tension, which might induce cell proliferation and migration, was suppressed during contractile differentiation. These results suggest that suppression of cell traction force and enhanced force polarity might be key factors in VSMC differentiation induced by low serum culture.
文摘Cells sense the external environment such as a surface topography and change many cellular functions. Cell nucleus has been proposed to act as a cellular mechanosensor, and the changes in nuclear shape possibly affect the functional regulation of cells. This study demonstrated a large-scale mechanical deformation of the intracellular nucleus using polydimethylsiloxane (PDMS)-based micropillar substrates and investigated the effects of nuclear deformation on migration, proliferation, and differentiation of vascular smooth muscle cells (VSMCs). VSMCs spread completely between the fibronectin-coated pillars, leading to strong deformations of their nuclei resulted in a significant inhibition of the cell migration. The proliferation and smooth muscle differentiation of VSMCs with deformed nuclei were dramatically inhibited on the micropillars. These results indicate that the inhibition of proliferation and VSMC differentiation resulted from deformation of the nucleus with high internal stress, and this type of large-scale nuclear mechanical stress might lead the cells to a “quiescent state”.
