Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.

Living cells respond to mechanical forces by changes in cellular structure and function through gene regulation and posttranslational protein modification (1–5). Reorganization of the cytoskeleton is a key component of the cellular response to mechanical stimuli (3, 6). The actin cytoskeleton is a dynamic filamentous network that determines cell shape and strength and plays an important role in cellular response to mechanical tension (7). Calponin is an actin filament-associated regulatory protein (8) and has been extensively studied for its role in the contractility of smooth muscle (9, 10). Biochemical activities of calponin have been documented in details mainly from experiments using the h1 isoform in smooth muscles (11). Through high affinity binding to F-actin, calponin inhibits the actin-activated smooth muscle myosin MgATPase and the production of force (12). Despite the extensive investigations, the physiological function of calponin in living cells remains to be established. The h2 isoform of calponin (11) is found in smooth muscle and non-muscle cells. Calponin's association with actin stress fibers and function in regulating actin-myosin interaction suggest a role in cytoskeleton activities. Forced expression of calponin in smooth muscle cells and fibroblasts inhibited cell proliferation (13, 14). Providing a novel lead for the function of calponin, we recently demonstrated a mechanical tension-regulated expression of h2-calponin in fibroblasts and epidermal keratinocytes with a role in stabilizing the actin filaments (15). In the responses of h2-calponin function to mechanical tension, gene regulation represents a chronic and sustained control. On the other hand, proteolysis may provide rapid structural and functional modifications during adaptations to environmental changes. Selective proteolysis can remove regulatory proteins when they are not needed, while transforming others from the dormant into the biological active state (16). Therefore, proteolytic regulation of calponin may also play a role in cytoskeleton responses to mechanical stimuli. In the present study, we examined the expression of h2-calponin in multiple representative tissues and found that it is abundant in lung alveolar epithelial cells. The lung undergoes dynamic mechanical tension changes from alternating distension and collapse that modulate the phenotypes of alveolar epithelial cells (17), and so is an informative system to study the tension-responsive regulation of cytoskeletal proteins. The expression of h2-calponin is rapidly up-regulated during postnatal lung development corresponding to respiratory expansion. In addition to the mechanical tension dependent expression and role in stabilizing actin cytoskeleton, a novel finding in the present study is that h2-calponin is regulated in alveolar cells by mechanical tension dependent proteolysis. A rapid degradation of h2-calponin occurs in lung tissues after prolonged deflation, which is effectively prevented at the inflated state. Decreasing mechanical tension in cultured alveolar cells by reducing the matrix dimension reproduced the degradation of h2-calponin. The myosin II ATPase-based tension in the actin cytoskeleton is required to prevent h2-calponin degradation. Another interesting finding is that continuous cyclic stretching of cells did not increase but decreased the expression of h2-calponin. Therefore, after the cellular structure is remodeled to fit the stretched dimension, cyclic relaxations would periodically release cytoskeleton tension and lower the total amounts of tension over time, which determines the expression of h2-calponin. The tension regulation of h2-calponin synthesis and degradation provides new insight into the mechanical function of lung alveolar cells that are physiologically under distension-relaxation stimuli and demonstrates a novel mechanical regulation of cellular biochemistry.