h2-calponin Is Regulated by Mechanical Tension and Modifies the Function of Actin Cytoskeleton

Abstract

Calponin is an extensively studied actin-binding protein, but its function is not well understood. Among three isoforms of calponin, h2-calponin is found in both smooth muscle and non-muscle cells. The present study demonstrates that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin. The expression of h2-calponin is cell anchorage-dependent. The levels of h2-calponin decrease when cells are rounded up and remain low when cells are prevented from adherence to a culture dish. h2-calponin expression resumes after the floating cells are allowed to form a monolayer in plastic dish. Cell cultures on polyacrylamide gels of different stiffness demonstrated that h2-calponin expression is affected by the mechanical properties of the culture matrix. When cells are cultured on soft gel that applies less traction force to the cell and, therefore, lower mechanical tension in the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Force-expression of h2-calponin enhanced the resistance of the actin filaments to cytochalasin B treatment. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Lowering the tension of actin cytoskeleton by inhibiting non-muscle myosin II ATPase decreased h2-calponin expression. In contrast to the mechanical tension regulation of endogenous h2-calponin, the expression of h2-calponin using a cytomegalovirus promotor was independent of the stiffness of culture matrix. The results suggest that h2-calponin represents a novel manifestation of mechanical tension responsive gene regulation that may modify cytoskeleton function.Calponin is a family of actin-associated proteins first found in smooth muscle cells (1). Three calponin isoforms (h1-, h2-, and acidic calponins) are encoded by three homologous genes. h1-calponin (2, 3) is specifically expressed in differentiated smooth muscle cells and has been extensively studied for its role in the regulation of smooth muscle contractility (for review, see Refs. 4 -6). The acidic calponin is found in nervous tissues and implicates in neuronal regeneration and growth (7-8).h2-calponin (3) is found in both smooth muscle and non-muscle cells such as human epidermal keratinocytes (9). h2-calponin mRNA has been also detected in endothelial cells (10) and fibroblasts (11). The gene regulation and function of h2-calponin are largely unknown. Previous studies suggested that h2-calponin may play a role in the organization of actin cytoskeleton (12) and in cytokinesis (13). This hypothesis is supported by the observation that h2-calponin is expressed at significant levels in growing and remodeling tissues (13). Forced expression of h2-calponin in cells lacking endogenous calponin results in an association with the actin stress fibers and a decrease in the rate of cell proliferation (13). These results suggest a microfilament-associated activity of h2-calponin, which may regulate the function of actin cytoskeleto...

Cytoskeletal Tension Regulates Both Expression and Degradation of h2-Calponin in Lung Alveolar Cells

“…Densitometry quantification of the h2-calponin Western blots against the levels of actin, total cellular protein, or histones ( Figure 11B) shows that cyclic stretching did not increase but significantly decreased h2-calponin expression by nearly 40%. This intriguing finding is actually consistent with the observation that h2-calponin expression is regulated by the tension generated in the cytoskeleton against the stiffness of cultural substrate (15). The cells cultured on the rubber membrane of the cyclic stretch plate have built up near maximum tension in the cytoskeleton close to that in cells cultured on a rigid (plastic) substrate since the silicon rubber membrane, same as the plastic dish, is non-deformable by the cellular contractile force.…”

“…The continuous vibration prevented cell attachment to the culture dish (23). The cells were harvested after 3 days of vibrating culture to examine the levels of h2-calponin by Western blotting as described previously (15). The floating cell aggregates were re-seeded on tissue culture dishes and incubated without vibration to confirm their viability.…”

“…The treated cells were washed with PBS and fixed with cold acetone. The actin stress fibers was examined by rhodamine-labeled phalloidin staining as described previously (15) and examined by fluorescence microscopy for the resistance to cytochalasin B destruction.…”

“…After 3 days of culture, the cells were treated with 100 μM blebbistatin, a myosin II ATPase inhibitor that decreases the myosin motorgenerated tension in cytoskeleton (27), as described previously (15,28) for either 6 hours or 3 days. Cells were cultured in normal media or media containing 0.2% DMSO (solvent of the blebbistatin stock) as controls.…”

“…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).…”