The intracellular mechanical link tethering the nucleus to the cytoskeleton has been suggested to be the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Previous studies have reported that knockdown of nesprin-1, a component of the LINC complex that directly binds to actin filaments, suppresses cellular morphological response to mechanical stimuli. The relation between nesprin-1 knockdown and cellular morphological changes, however, remains unclear. In this study, we examined the mechanical role of nucleus-actin filament binding in morphological changes of fibroblasts exposed to cyclic stretching. After exposure to 10% cyclic stretching for 6 h, fibroblasts transfected with nesprin-1-specific small interfering RNA showed fewer elongated shapes compared with non-transfected cells. To further examine the mechanical role of the nucleus and nucleus-bound actin filaments, we applied cyclic stretching to fibroblasts treated with Trichostatin A (TSA), which decreases nuclear stiffness and thereby reduces nucleus-binding actin filament tension. TSA-treatment was found to induce more rounded cellular shapes than those of non-treated cells under both static and cyclic stretching conditions. These results suggest that the tension of nucleusbound actin filaments plays an important role in the formation of elongated fibroblasts under cyclic stretching and that nesprin-1 knockdown causes a decrease of tension in nucleus-associated actin filaments.

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Macrophages infiltrated in the walls of abdominal aortic aneurysms (AAA) are subjected to cyclic stretching due to pulsatile deformation of blood vessels and low oxygen conditions caused by intraluminal thrombus. These conditions could induce aberrant changes in macrophage functions and lead local weakening of AAA walls. We previously reported that the combination of 10 % cyclic stretching and 2.2% O 2 conditions caused an increase in matrix metalloproteinase-9 (MMP-9) production in macrophages as well as changes in morphological responses to cyclic stretching. In the present study, we investigated the effects of oxygen concentrations on MMP-9 productions and morphological changes of macrophages subjected to cyclic stretching. Macrophages differentiated from THP-1 cells were subjected to 10% cyclic stretching under 5% or 1% O 2 conditions for 24 h, and gelatinolytic activity of MMP-9 in the conditioned medium was assessed by zymography. Cells showed spread and rounded shape under static conditions and elongated and oriented to the direction of stretching after exposure to cyclic stretching, and there were no obvious effects of oxygen concentrations in cell morphology. An O 2 concentration of 5% did not change MMP-9 productions of macrophages in static culture and subjected to cyclic stretching compared to normal cell culture condition of 20% O 2 . In contrast, 1% O 2 condition stimulated MMP-9 production of macrophages both under static culture and cyclic stretching conditions. We also found that treatments of inhibitors for extracellular signal-regulated kinase (ERK) and Rho associated protein kinase (Rho kinase) suppressed the increased MMP-9 productions of macrophages by 1% O 2 condition. These results suggest that lower oxygen conditions such as 1% O 2 stimulate MMP-9 production in macrophages through signaling pathways involving ERK as well as Rho kinase-mediated actin cytoskeletal contractility.

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