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.

Fig. 1.

H2-calponin expression in epidermal keratinocytes. (A) Total protein extracts from mouse abdominal and footpad skin tissues were analyzed by Western blots using the anti-h2-calponin polyclonal antibody RAH2 and anti-h1-calponin mAb CP1. Purified mouse h1- and h2-calponin proteins were used as control. Normalized by the level of actin, the blots show a significant level of h2-, but not h1-, calponin in the mouse skin, especially footpad. (B) Thin paraffin sections of human epidermal scar tissues were examined by immunocytochemistry with anti-h2-calponin mAb 1D2 and SP2/0 myeloma cultural supernatant control. The results show h2-calponin expression in the keratinocyte layers. (C) Western blots using anti-h2-calponin polyclonal antibody RAH2 and anti-h1-calponin mAb CP1 on total protein extracts from human keratinocytes cultured 3 days on plastic dish detected high levels of h2- but not h1-calponin. The sample loading was normalized by the level of actin and purified mouse h1- and h2-calponins were included as control.

Fig. 2.

Culture time-dependent expression of h2-calponin in keratinocytes. From three-day old pre-confluence cultures, human epidermal keratinocytes were passed on plastic dishes at low or high densities. The cells were harvested at 1, 2, 3, 4 and 5 days after plating and the total protein extracts were examined by SDS-PAGE and Western blot using anti-h2-calponin polyclonal antibody RAH2 (A). Normalized against the actin band in the accompanying SDS-gel, densitometry quantification of the Western blots was used to compare the levels of h2-calponin expression (B). The results show that independent of the high (solid column) or low (open column) cell densities (the cells were at 100% and 60% confluence at three days in culture, respectively), the expression of h2-calponin was low at one day after plating (P < 0.001) and returned to the maximum level three days after plating. The results are summarized from 6 individual experiments.

Fig. 3.

Matrix anchorage-dependent expression of h2-calponin in keratinocytes. Human epidermal keratinocytes were cultured on plastic cultural dishes steadily as a monolayer or with continuous vibration that prevented the cells from anchoring on the dish. After 3 days of culture, the cells were photographed for phase contrast images (A) and harvested to examine the total protein extracts by Western blotting with anti-h2-calponin polyclonal antibody RAH2 (B). Normalized by the actin level, the results show that the floating keratinocytes growing in multi-cell aggregates had significantly reduced h2-calponin expression. Similarly, phase contrast images (C) show continuous vibration prevented KD human fibroblasts from anchoring to the cultural dish and produced multi-cell aggregates. When the floating cell aggregates were re-seeded on plastic dish and cultured without vibration, they attached to the dish to form a monolayer similar to the steady culture control of the same age. The result verifies the viability of the cells in the floating aggregates. (D) Total protein extracts from the KD cells were examined by SDS-PAGE and Western blotting using anti-h2-calponin antibody RAH2. Normalized by the level of actin, the results show that KD cells lost h2-calponin expression when growing as floating aggregates. The expression of h2-calponin resumed when the floating cells anchored to the cultural dish.

Fig. 4.

Matrix stiffness dependent expression of h2-calponin in keratinocytes. Keratinocytes were plated on plastic surface or polyacrylamide gels of different stiffness. After 3 days of culture, the levels of h2-calponin were determined by Western blot analysis using RAH2 antibody. Normalized by the amount of actin, densitometry quantification results in show a significantly lower expression of h2-calponin in keratinocytes grown on soft polyacrylamide gel in comparison with that of the hard gel and plastic dish cultures (^* P < 0.001). The results were summarized from 5 individual experiments.

Fig. 5.

Cell spreading areas when cultured on matrices of different stiffness. Keratinocytes were cultured on plastic surface or polyacrylamide gels of different stiffness. (A) Phase contrast images at 3 days of culture show that the cells attached and grew in monolayers on the plastic and gel matrices. The cell spreading areas 4 hr after plating were significantly lower (^* P < 0.001) in the soft gel culture as compared with the very hard gel and plastic controls (B). However, the cell spreading areas under the three matrix stiffness conditions increased at similar rates during culture (C).

Fig. 6.

Increased h2-calponin expression during Ca²⁺-induced differentiation of keratinocytes grown on soft matrix. Human epidermal keratinocytes were plated on soft polyacrylamide gel or plastic dish and induced with 0.3mM CaCl₂ at 40-70 % confluence for 48 hours. (A) Phase contrast images show that in contrast to non-induced spreading cell culture, the cells in both soft and rigid matrix cultures migrated together upon Ca²⁺ induction. (B) Normalized by the levels of actin, total protein extracted from the cells was analyzed by Western blot to examine the expression of h2-calponin and involucrin. The increase in involucrin level upon Ca²⁺ induction verified the differentiation of keratinocytes in cultures on soft and rigid matrix. While the expression of h2-calponin was very low in keratinocytes cultured on soft gel, it is significantly up-regulated upon Ca²⁺ induction (^* P < 0.001). Different from the soft matrix cultures, the expression of h2-calponin in the cells cultured on rigid matrix was already high and did not further increase during differentiation. Five individual experiments each were performed for the soft and rigid matrix differentiation experiments.

Fig. 7.

Association of h2-calponin with actin cytoskeleton. Human keratinocytes and KD fibroblasts were cultured on gelatin-coated glass cover slips. (A) Pre-confluent monolayer keratinocyte cultures were examined by immunofluorescence microscopy with the rabbit anti-h2-calponin antibody RAH2 or anti-tropomyosin (hTM4) mAb LC24 in comparison with TRITC-phalloidin stained actin filaments. (B) Immunofluorescence assay on KD fibroblast culture was carried out using anti-h2-calponin antibody RAH2 and anti-tropomyosin (hTM5) mAb CG3 together with TRITC-phalloidin control. (C) Confocal microscopy of the double-stained KD cells demonstrates the co-localization of h2-calponin (green) and Tm (red) in actin stress fibers (the yellow color). Plotting the intensity of co-localized h2-calponin and Tm stains (right panel) shows a positive correlation.

Fig. 8.

H2-calponin stabilizes actin cytoskeleton. (A) Three-day old monolayer cultures of h2-sense and antisense cDNA stable transfected SM3 cells were treated with serial concentrations of cytochalasin B (0.5, 0.75 and 1.0 μg/ml) at 37°C for 30 minutes. The cells were then fixed with cold acetone and actin stress fibers were visualized by staining with TRITC-conjugated phalloidin. The fluorescence microscopic images show that the presence of h2-calponin produced a higher tolerance of the actin cytoskeleton to cytochalasin B. (B) A significant level of h2-calponin was expressed in the sense cDNA transfected SM3 cells as shown by the RAH2 Western blot in contrast to the non-transfected and antisense cDNA transfected controls. Normalized by the level of actin in SDS-gel, Western blots using a mixture of mAbs CGβ6, LC24 and CG3 showed that the level of tropomyosin (hTm2 + hTM3 + hTM4 + hTM5) was higher in the sense h2-calponin cDNA transfectedSM3 cells than that in antisense and non-transfected controls. (C) Correlation analysis on three original h2-calponin sense cDNA-transfected SM3 cell lines showed a positive relationship between the levels of h2-calponin and tropomyosin (P < 0.05).

Fig. 9.

Myosin II inhibitor reduces h2-calponin in NIH 3T3 cells. (A) Western blots using RAH2 antibody detected a significant amount of h2, but not h1, calponin in NIH 3T3 mouse fibroblast, comparable with that in human epidermal keratinocytes and KD fibroblasts. The results were confirmed by Western blots using ant-h2-calponin mAb 2B8 (13) and anti-h1-calponin mAb CP1 (39). The protein loading was normalized by the level of actin. Purified human h2-, mouse h1- and h2-calponins were used as controls. (B) Three-day old monolayer 3T3 cell cultures on plastic dishes or gelatin-coated cover slips were treated with 100 μM blebbistatin for 3 days. Phase contrast images of the cells were taken before fixation. The cells on the cover slips were examined for actin stress fibers by TRITC-conjugated phalloidin staining after acetone fixation. The results show that the blebbistatin treated cells became slack with diminished actin stress fibers. (C) H2-calponin levels were determined by Western blot analysis with RAH2 antibody. Normalized by the amount of actin, densitometry quantification results show a significantly decrease in h2-calponin in blebbistatin-treated cells in comparison with that of non-treated (P < 0.01) and DMSO (P < 0.001) controls. An increase in h2-calponin in the DMSO control was observed (P < 0.01). The results were summarized from 4 individual experiments.

Fig. 10.

Mechanical tension independent expression of viral promoter-directed h2-calponin transgene. (A) Time course of h2-calponin levels in h2-calponin sense cDNA transfected SM3 cells after passage. The results are summarized from densitometry of multiple Western blots using RAH2 antibody on total protein extracts from two original stable transfected cell lines. In contrast to the post cell roundup decrease of endogenous h2-calpon in keratinocytes and fibroblasts, the level of CMV promoter force-expressed h2-calponin in SM3 cells was not decreased but slightly increased at 12 and 24 h after cell passage (P < 0.01). (B) H2-calponin sense cDNA transfected SM3 cells were cultured on plastic dishes with or without continuously vibration that prevented cell attachment on the dish. Three days old floating cells were further cultured without vibration to form a monolayer. The cells were harvested at each of the states for Western blot analysis using RAH2 antibody to examine the levels of h2-calponin. The sample loading was normalized by their actin contents. Densitometry quantification of the blots shows no decrease but an increase in h2-calponin in the floating cells (^*P < 0.05). (C) Transfected SM3 cells cultured for 3 days on plastic dishes or soft polyacrylamide gels were examined by SDS-gel and Western blot using RAH2 antibody for the level of h2-calponin. Quantified by densitometry and normalized by the actin bands on parallel gel, the results showed a higher level of h2-calponin in the soft gel cultures than that of the plastic dish control (^*P < 0.001). The results for the cell anchorage and matrix stiffness experiments were each summarized from 4 individual experiments.