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J. Biol. Chem., Vol. 280, Issue 51, 42442-42453, December 23, 2005
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1
From the
Section of Molecular Cardiology, Evanston Northwestern Healthcare and Northwestern University Fienberg School of Medicine, Evanston, Illinois 60201, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, and ¶Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
Received for publication, September 9, 2005 , and in revised form, October 12, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONREFERENCES |
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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 cytoskeleton.
The actin cytoskeleton has essential functions in many cellular activities, such as cell division (14), migration (15), and contraction (16). A major component of the actin filaments in higher eukaryotes is tropomyosin. Tropomyosin is known to participate in the regulation of striated muscle contraction (17) and to stabilize actin filaments in non-muscle (18) as well as muscle cells (19). Calponin binds actin and tropomyosin and is proposed to be an analog of striated muscle troponin (1, 20) and, therefore, may regulate the actin-myosin II-based cytoskeleton function.
Previous studies have shown that living cells respond to mechanical forces exerted through surrounding fluid, adhered beads, or cultural matrix (21-25). Physical properties of the adhesion substrate can profoundly affect cell locomotion, growth, and differentiation (26-28). The stiffness of the cultural matrix has effects on cell motility (29, 30), phagocytosis (31), and differentiation (32, 33). Varying stiffness of the cultural matrix applies different traction force to the cell and, therefore, different mechanical tension to the cytoskeleton (34, 35). The regulation and function of actin cytoskeleton in the cellular responses to mechanical tension represents an important but little known subject of signal transduction.
In the present study we investigated the regulation of h2-calponin by mechanical tension. We demonstrated that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin, which decrease when cells are rounded up and remain low when cells are prevented from anchorage on the culture dish. h2-calponin expression resumes after the floating cells are allowed to attach to the plastic dish. When cells are cultured on soft gel matrix that applies lower mechanical tension to the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Force-expression of h2-calponin increased actin filaments resistance to cytochalasin B treatment. 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 (CMV)2 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.
Specific Antibodies—A rabbit polyclonal antiserum (RAH2) raised against mouse h2-calponin with a weak cross-reaction to h1-calponin has been described previously (36). Monoclonal antibody (mAb) 1D2 against human h2-calponin was developed as described previously for the production of anti-mouse h2-calponin mAbs (13). Briefly, human h2-calponin cDNA was cloned using reverse transcription-coupled polymerase chain reaction as described previously (36) and expressed in Escherichia coli for protein preparation (37). Purified human h2-calponin was used to immunize 8-week-old female Balb/c mice in a short-term immunization protocol (38). Spleen cells were harvested from the immunized mouse for fusion with SP2/0-Ag14 mouse myeloma cells. Hybridoma colonies were screened by indirect enzyme-linked immunosorbant assay and subcloned three times to establish a stable cell line. The mAbs were produced in the forms of hybridoma cultural supernatant and mouse ascites fluids. Enzyme-linked immunosorbant assay immunoglobulin isotyping (Invitrogen) determined its subclass as IgG2b
. Its specificity was determined by Western blotting on purified h1- and h2-calponins (37).
Mouse anti-h1-calponin mAb CP1 was prepared and characterized previously (39). Mouse anti-tropomyosin isoform mAbs, CG1 against hTM1, CG
6 against hTM2 and hTM3, LC24 against hTM4, and CG3 against hTM5 were described previously (40-43). A rabbit anti-human involucrin antibody was produced as previously described (44, 45).
Western Blotting—PAGE and Western blotting were carried out as described previously (38) to examine the expression of calponin in mouse skin tissues and human keratinocyte and fibroblast cultures. The samples were homogenized in SDS gel electrophoresis sample buffer containing 2% SDS and analyzed on 12% gel with an acrylamide:bisacrylamide ratio of 29:1 in the Laemmli buffer system. After electrophoresis, the gels were fixed and stained with Coomassie Blue R250 to confirm sample integrity and protein contents. Protein bands in unfixed duplicate gels were transferred to nitrocellulose membrane for Western blotting with the anti-calponin, anti-tropomyosin, or anti-involucrin antibodies. The specific protein bands recognized by the first antibodies were revealed by using alkaline phosphatase-labeled anti-rabbit IgG or anti-mouse IgG second antibody (Sigma) and 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chromogenic substrate reaction. Purified h2- and h1-calponins (37) was used as control.
Immunohistochemistry—To examine the in vivo expression of h2-calponin in epidermal keratinocytes, paraffin sections of epidermal scar (anonymously provided by the Skin Disease Research Center at Case Western Reserve University School of Medicine) were stained with anti-h2 calponin mAb 1D2 that had no cross-reaction to h1-calponin followed by horseradish peroxidase-labeled anti-mouse IgG second antibody (Sigma) and H2O2/3,3'-diamino-benzidine substrate reaction using standard immunohistochemical methods (46). To avoid background staining of tissue sections by immunosera and mouse ascites fluid (data not shown), 1D2 hybridoma culture supernatant was used. The morphology of tissue sections was outlined by counterstaining of the slides with 0.6% hematoxylin for 20 s.
Cell Cultures—Primary keratinocytes were isolated from human foreskin samples (47) and cultured in serum-free keratinocyte media (Invitrogen) with supplements (50 µg/ml bovine pituitary extract, 5 ng/ml human recombinant epidermal growth factor; Invitrogen) at 37 °C in 5% CO2. Culture media were replaced every 3 days. The monolayer cells around 70% confluence were passed at 1:5 ratio, and cells at second or third passages were used for experiments. The methods for culturing human epidermal keratinocytes were described in detail previously (48, 49).
Human Fibroblast Cell Line KD (American Type Culture Collection, CRL 1295) was cultured in Dulbecco's-modified Eagle medium containing 10% fetal bovine serum, penicillin (100 IU/ml) and streptomycin (50 IU/ml) at 37 °C in 5% CO2.
SM3 is an immortalized cell line derived from rabbit aortic smooth muscle (50) and has ceased endogenous calponin expression (13). Stable transfected SM3 cells force-expressing h2-calponin driven by the CMV promoter in pcDNA3.1 vector (13) were used in the present study to investigate h2-calponin regulation and functional effect. The sense cDNA construct expresses non-fusion full-length mouse h2-calponin protein for authentic functional characterization, and antisense cDNA-transfected cell lines were used as negative control. The transfected SM3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 IU/ml), streptomycin (50 IU/ml), and G418 (100 µg/ml) at 37 °C in 5% CO2.
Immunofluorescence Microscopy—Precleaned glass coverslips were coated with 0.1% gelatin and dried under UV radiation for 3 h before being placed in the culture dish. Keratinocytes and KD cells were seeded to grow a monolayer on the coverslips. After 3 days of culture, the cells grown on coverslips were washed with Dulbecco's phosphate-buffered saline (D-PBS; 136.89 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2). The cells were then fixed with cold acetone for 20 min. Immunofluorescence microscopy was carried out as described previously (13) to examine the cellular localization of h2-calponin and tropomyosin using anti-calponin and anti-tropomyosin antibodies. TRITC- or fluorescein isothiocyanate-conjugated goat anti-rabbit or anti-mouse IgG second antibodies (both from Sigma) were used to distinguish the rabbit and mouse first antibody staining representing the localization of calponin and tropomyosin, respectively, using a Zeiss Axiovert 100H phase contrast-epifluorescence microscope with two sets of filters (CZ915 and CZ909). Actin stress fibers in the cell were examined by rhodamine-labeled phalloidin (Sigma) staining. The co-localization of h2-calponin and tropomyosin in KD cells were also examined by confocal microscopy at the Neurobiology Core Facility at Case Western Reserve University School of Medicine.
Examination of Cell Plating Time Dependence of h2-calponin Expression—To investigate the expression of h2-calponin during the time course of monolayer cell culture, preconfluent keratinocytes were trypsinized and seeded at high and low densities (initial density = 9 x 105 cells and 3 x 105 cells/60-mm dish, respectively). The cells were suspended from the culture dishes after 1, 2, 3, 4, and 5 days using the Versene solution (0.537 mM EDTA, 136.8 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, pH 7.2) and washed 3 times with D-PBS. Omitting trypsin digestion during the cell harvest avoids artificial degradation of cellular proteins. SDS gel sample buffer containing 2% SDS was used to lyse the cells, and total cellular protein was extracted by vortexing. Levels of h2-calponin in high and low density keratinocyte cultures at the post-plating time points were examined by Western blot analysis using anti-h2-calponin antibody RAH2. KD fibroblasts and stable transfected SM3 cells were examined similarly for the plating time effect on the levels of h2-calponin.
Examination of Matrix Adhesion-dependent Expression of h2-calponin—To examine the effects of matrix attachment on the expression of h2-calponin in cultured cells, continuous vibration was applied to keratinocytes, KD fibroblasts, and SM3 cells to prevent cell attachment to tissue culture dishes (51). Incubated on a humidified CO2 incubator at 37 °C, the cultural dishes were placed on an orbital shaker (Bel-Art Products, Pequannock, NJ) driven by a magnetic stirrer at 80 rpm. The cells were harvested after 3 days of culture in vibration to examine the levels of h2-calponin. After washed with PBS, the cells were lysed in SDS-gel sample buffer and examined by Western blotting analysis as described above. The viability of the floating cell aggregates was confirmed by growing into monolayers after re-seeding on tissue culture dishes and incubated without vibration. The expression of h2-calponin in the cells after re-seeding was examined as described above.
Examination of Culture Matrix Stiffness-dependent Expression of h2-calponin—To compare with the rigid plastic cultural dish, a thin layer of polyacrylamide gel was prepared on glass coverslips to provide a soft matrix for cell culture as described previously (30, 35, 52). The coverslips were passed briefly through the inner flame of a Bunsen burner. A drop of 0.1 N NaOH was smeared across the surface of the coverslip and air-dried. Thereafter, the coverslips were treated with 3-aminopropyltrimethoxysilane and then with 0.5% glutaraldehyde. Polyacrylamide gels of different stiffness (very hard, 10% gel with an acrylamide:bisacrylamide ratio of 40:1; hard, 3% gel with an acrylamide: bisacrylamide ratio of 14:1; soft, 3% gel with an acrylamide:bisacrylamide ratio of 74:1) were polymerized in between the treated coverslip at the bottom and an untreated coverslip on the top. After polymerization, the untreated coverslip was removed to expose the 
100-µm thin layer of gel to be used as cell culture matrix. Previous studies have shown that the stiffness of the polyacrylamide gel matrix is directly proportional to the concentration of bisacrylamide cross-linker (30, 34, 52).
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To examine the effect of the tension that the culture matrix applied to the cell through traction force on the expression of h2-calponin, keratinocytes were seeded on a plastic culture dish or polyacrylamide gels of different stiffness. The cells were harvested after 3 days of culture by directly lysis in SDS gel sample buffer after D-PBS washes. The level of h2-calponin was examined by Western blot analysis with the anti-h2-calponin antibody RAH2 as described.
Measurement of Cell Spreading Area—Monolayer keratinocytes cultured on plastic dish or polyacrylamide gels of different stiffness were photographed at 4, 12, 24, 48, and 72 h of culture. The NIH Image program Version 1.61 was used to measure the two-dimensional spreading area of randomly selected non-overlapping cells.
Ca2+ Induction of Keratinocyte Differentiation—To examine the expression of h2-calponin during keratinocyte differentiation in vitro, cells were plated on plastic culture dishes or soft polyacrylamide gel (3% with an acrylamide:bisacrylamide ratio of 74:1). One day after plating, the concentration of CaCl2 in culture media was increased to 0.3 mM. The cells were harvested after 48 h in the high Ca2+ media for Western blotting examination of h2-calponin as described above. The expression of involucrin, an established cell differentiation marker, in epidermal keratinocytes was examined by Western blot to monitor the induction of differentiation (53-55).
Cytochalasin B Treatment—To investigate the effect of h2-calponin on actin cytoskeleton, stable transfected SM3 cells expressing h2-calponin were plated on gelatin-coated coverslips. After 3 days of culture, the cells were treated with 0.5, 0.75, and 1.0 µg/ml cytochalasin B (Sigma) at 37 °C for 30 min. h2-calponin-negative SM3 cells were examined as the control. The cytochalasin B stock was prepared in Me2SO, so a 0.2% Me2SO-treated control group of cells was also examined. The cells were washed with PBS and fixed with cold acetone. The structure of stress fibers stained with rhodamine-labeled phalloidin was examined as above and evaluated for the resistance to cytochalasin B treatment.
Blebbistatin Treatment—To examine the effect of reducing cytoskeleton tension, which is mainly built up by myosin II motor activity on the expression of h2-calponin in cells, NIH 3T3 cells were cultured on plastic dish and gelatin-coated coverslips (3 x 104 cells/35-mm dish). After 3 days of monolayer culture, the cells were treated with 100 µM blebbistatin (Sigma), a myosin II ATPase inhibitor (56), for 3 days. Parallel cultures in normal media or containing 0.2% Me2SO (solvent for making the blebbistatin stock) were examined as controls. The cells in culture dish were harvested by lysis in SDS-PAGE sample buffer after PBS washes, and the levels of h2-calponin were determined by Western blot analysis as above. The cells on the coverslips were fixed with cold acetone, and the actin stress fibers were stained with rhodamine-conjugated phalloidin. The fluorescence and phase contrast images were viewed under a Zeiss Axiovert 100H fluorescence microscope.
Data Analysis—Densitometry analysis of SDS-gel and Western blots was done on digital images scanned at 600 dots/inch, and the NIH Image program Version 1.61 was used to quantify the levels of h2-calponin and tropomyosin expression. The calponin and tropomyosin bands detected in Western blots were normalized by the amounts of actin in equally loaded SDS gel. The quantitative data of the cell area, h2-calponin, and tropomyosin levels are presented as the mean ± S.D. Statistical analysis was done using the Microsoft Excel computer program.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONREFERENCES |
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Expression of h2-calponin Is Dependent on Cell Anchorage to the Culture Dish—Two factors may confer the recover of h2-calponin expression during the 3 days of culture after cell passage. One is the culture time after passage, and the other is the re-attachment to the culture dish. We investigated which factor determines h2-calponin expression by producing floating cell cultures. The results in Fig. 3, A and B, show that when keratinocytes were prevented from attachment to the culture dish by continuous vibration, the cells grew in multicell aggregates with significantly decreased h2-calponin expression.
Because primary human epidermal keratinocytes may not survive well in the floating culture, the observed phenotype after passages often vary between experiments. We, therefore, verified this observation using the human fibroblast cell line KD. The Western blots in Fig. 3, C and D, show a significant expression of h2-calponin in the KD fibroblast monolayer cultured on plastic dishes. When KD cells were cultured with continuous vibration, they also grow in multi-cell aggregates (Fig. 3C). Western blot analysis showed that the floating KD cells completely lost h2-calponin, in sharp contrast to the high level expression in monolayer cells attached on plastic dish (Fig. 3D). After the floating cells were re-plated and cultured without vibration, they attached to the plastic dishes and regained high level expression of h2-calponin (Fig. 3D). The results verify the viability of the floating cells cultured under vibration and further demonstrate that h2-calponin expression is dependent on the anchorage to the plastic culture dish other than the culture time after passage. Consistent with the independence of cell-cell contacts as shown in the cell density comparisons (Fig. 2), the aggregation of floating cells does not induce h2-calponin expression.
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It has been established that centripetal-directed traction forces exerted on cells and cell areas are increased with increasing stiffness of culture matrix (29). The spreading area of keratinocytes at 4 h after plating was smaller when cultured on soft gel than that of the very hard gel or plastic controls (Fig. 5, A and B). The less spreading morphology of the cells cultured on a soft matrix implies that less tension is applied to the cytoskeleton than that in the more spreading cells cultured on hard matrices. Nonetheless, the cell area increases at similar rates during culture under these conditions (Fig. 5C), indicating adjusted cytoskeleton activity.
Up-regulation of h2-calponin during Ca2+-induced Differentiation of Keratinocytes—Keratinocyte is the major cell type of the continually renewing epidermis and undergoes a choreographed program of differentiation. An increase in extracellular Ca2+ levels is thought to induce the keratinocyte differentiation in the skin (57). To evaluate the functional significance of mechanical tension-related expression of h2-calponin in epidermal keratinocytes, its relationship with phenotypic differentiation was investigated. The results in Fig. 6 show that when keratinocytes were cultured on a soft gel matrix, Ca2+ induced differentiation as shown by the expression of involucrin. The low level expression of h2-calponin on the soft matrix was significantly increased during differentiation, suggesting a role in the phenotype transition. In contrast, keratinocytes grown on plastic matrix already had a high level expression of h2-calponin before differentiation, which did not show a further increase after differentiation (Fig. 6). The observation that the Ca2+-induced differentiation and high mechanical tension did not have additive effects on the level of h2-calponin suggests that h2-calponin gene regulation is a common target of these two very distinct cell signaling mechanisms.
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h2-calponin Increases Actin Filament Resistance to Cytochalasin B—By binding to the barbed ends of actin filament and, thus, inhibiting polymerization, cytochalasins disrupt actin cytoskeleton and can be used to evaluate the stability of actin filaments (58). We investigated the role of h2-calponin in the function of actin cytoskeleton by examining the effects of cytochalasin B on actin stress fibers. SM3 cell provides an endogenous h2-calponin null background to investigate the functional effect of transfective expression of h2-calponin. Normalized against actin, the h2-calponin contents in stable transfected SM3 lines, keratinocytes, and mouse and human fibroblast cell lines showed a wide range of variance (up to 2.4-fold differences, data not shown) where the levels of h2-calponin expressed in the transfected SM3 cells were within this natural range. The rhodamine-labeled phalloidin stain in Fig. 8A shows that the actin stress fibers in h2-expressing SM3 cells tolerated higher concentrations (>0.75 µg/ml) of cytochalasin B than that of the calponin null control SM3 cells. Tropomyosin has been demonstrated to contribute to the stability of actin filament in cytochalasin assays (59). Four isoforms of tropomyosin are detected in SM3 cells (hTM2, hTM3, hTM4, and hTM5), and all of them were increased in the presence of forced expression of h2-calponin (Fig. 8, B and C). Moreover, the increases in tropomyosin parallel the force-expressed levels of h2-calponin (r2 = 0.888, p < 0.05). These results suggest that h2-calponin may regulate the dynamics and function of actin filaments together with the function of tropomyosin. It is worth noting that the tropomyosin contents in non-transfected SM3 cells, keratinocytes, and KD and 3T3 fibroblast cell lines vary significantly (up to 3-fold differences; data not shown). Therefore, it is not simply the level of Tm that confers the functional effect of h2-calponin. The contribution of Tm to the cell phenotype changes corresponding the presence or absence of h2-calponin remains to be investigated.
Inhibition of Myosin II Results in Decreases in h2-calponin—Blebbistatin is a small molecule inhibitor that selectively binds non-muscle myosin II with high affinity, slows down its ATPase rate, and blocks myosin II in an actin-detached state (60). Blebbistatin provides a tool to reduce mechanical tension built into the actin cytoskeleton (61). When 3-day-old monolayer cultures of NIH 3T3 fibroblasts that express significant levels of h2-calponin (Fig. 9A) were treated with 100 µM blebbistatin, the morphology of the cells started to change within 1 h and remained at a "slack" state (Fig. 9B). h2-calponin expression was determined by Western blot analysis with anti-h2-calponin antibody RAH2. Densitometry quantification results show a significantly lower level of h2-calponin in blebbistatin-treated cells in comparison with that of non-treated (p < 0.01) and 0.2% Me2SO (p < 0.001)-treated cells (Fig. 9C). h2-calponin expression in Me2SO-treated cells was slightly increased (p < 0.01) in comparison to that in non-treated NIH 3T3 cells, suggesting that Me2SO may induce h2-calponin expression. Nonetheless, the opposite change indicates that the trace amount of Me2SO carried over from the blebbistatin stock is not responsible for the decrease in h2-calponin in the blebbistatin-treated group. The results support the hypothesis that mechanical tension in the actin filaments regulates h2-calponin expression. This mechanism is plausible for calponin function in regulating actin-myosin interaction and worth further investigation.
Viral Promoter-directed Expression of h2-calponin Is Independent of Cell Attachment or Matrix Stiffness—The regulation of h2-calponin by mechanical tension applied to the actin cytoskeleton and the role of h2-calponin in stabilizing actin filaments suggested two possible mechanisms of cellular regulation. One is that the mechanical signals regulate the expression of h2-calponin gene. The other is that a reorganization of cytoskeleton in response to mechanical tension alters the accumulation of h2-calponin. To investigate these potential mechanisms, we examined forced expression of h2-calponin by a viral promoter in SM3 cells, an immortalized cell line derived from rabbit aortic smooth muscle that has lost endogenous calponin expression. With a calponin-positive origin implying a compatible cellular environment, SM3 cell provides a calponin null background for investigating the function of exogenous h2-calponin. CMV promoter produced a high level expression of h2-calponin in monolayer SM3 cultures. The results in Fig. 10A show that in contrast to the post-cell roundup decreases of endogenous h2-calponin seen in keratinocytes (Fig. 2) and fibroblasts (data not shown), 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).
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Consistently, when the transfected SM3 cells were cultured on soft gel matrix, the level of h2-calponin did not decrease (Fig. 10C). Instead, as in the floating culture, there was an increase in h2-calponin level in cultures on the soft gel matrix. These results further support that the mechanical tension-dependent expression of endogenous h2-calponin in keratinocytes and fibroblasts is via gene regulation. On the other hand, the low tension-caused increase in h2-calponin expressed by the presumably mechanical tension-independent CMV promoter may reflect an increased accumulation of calponin protein in the actin filaments due to cytoskeleton reorganization, in agreement with h2-calponin proposed function in cytoskeleton remodeling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONREFERENCES |
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An established activity of h1-calponin is the inhibition of smooth muscle actomyosin ATPase (4-6). Structural conservation suggests that h1, h2, and acidic calponins may function via similar molecular mechanisms to regulate actin-myosin interactions. We previously showed that forced expression of h2-calponin in SM3 cells inhibits cytokinesis and the rate of cell proliferation (13). The association of h2-calponin to the actin filament in keratinocytes and fibroblasts (Fig. 7) is consistent with a role in the regulation of actin-based cell motility and other cellular activities (12). Similar to the fact that smooth muscle can contract normally without h1-calponin (36, 63, 64), not all tissues or cultured cells express h2-calponin. Therefore, h2-calponin seems not to be an essential protein for epithelial cells or fibroblasts to grow and function but may represent a regulatory mechanism that fine-tunes the function of actin cytoskeleton.
Mechanical Tension Regulates h2-calponin Gene Expression—Living cells can respond to both the magnitude and distribution of adhesion force (65). In the present study we demonstrated that the expression of h2-calponin in cell cultures is determined by cell anchorage to the culture matrix other than cell-cell contacts. The fact that the floating culture cells by continuously shaking do not induce h2-calponin expression (Figs. 3) suggests that the regulation is different from the fluid shear stress-related cell signaling (66). The down-regulation of h2-calponin in soft gel cultures is similar to that in floating keratinocytes. Although the two culture conditions are rather different, a common effect on the cytoskeleton is a lowered mechanical tension in comparison to the monolayer cells anchored on rigid culture matrix. Therefore, the similar down-regulation of h2-calponin under these two conditions suggests that mechanical tension is the key signaling mechanism that determines h2-calponin gene expression.
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In contrast to the endogenous h2-calponin in keratinocyte and fibroblasts, no post-passage transient decrease in levels was found for the transfective-expressed h2-calponin in SM3 cells (Fig. 10A). The viral promoter-driven expression of h2-calponin was independent of matrix stiffness (Fig. 10, B and C), indicating a primary control at the level of gene regulation. However, the decrease in h2-calponin levels after cell round-up during passages is a rapid process (data not shown). Proteolytic degradation may play a critical role in this process. The lack of post-round-up decreases of h2-calponin in SM3 cells suggests that the proteolytic mechanism is not a ubiquitous mechanism in all cells, and the mechanisms remain to be investigated. Although keratinocytes and KD fibroblasts reattach to the culture dish within 30 min after re-plating, 3 days are needed for their h2-calponin level to return to the high level before the passage. This slow recovery suggests that rapid degradation may have removed most of the h2-calponin protein, and de novo synthesis in response to high mechanical tension is required to restore h2-calponin levels in the cell. The lack of rapid degradation of exogenous h2-calponin in SM3 cells indicates that the proteolytic regulation may be cell type-specific.
Up-regulation of h2-calponin during Epidermal Keratinocyte Differentiation—Increases in extracellular Ca2+ concentration result in termination of cell growth and induction of a terminal differentiation phenotype of keratinocytes in culture (57, 67). An increasing gradient of Ca2+ concentration is present from the basal to the cornified layers of epidermis in the skin (68). The expression of h2-calponin in Ca2+-induced keratinocytes is significantly up-regulated when cells were cultured on soft gel matrix (Fig. 6). Although the keratinocytes grown on a plastic dish had high levels of h2-calponin expression that did not further increase upon differentiation, the epidermal keratinocytes in vivo do not reside on such a rigid matrix, and their anchoring environment is soft tissue, which the soft gel matrix may resemble. Our previous study showed that h2-calponin is expressed at higher levels in growing and remodeling tissues, whereas overexpression of h2-calponin inhibited cytokinesis and resulted in decreases in the rate of cell proliferation (13). The hypothesis is that the more rapidly remodeling cytoskeleton may need a more active h2-calponin regulation. Therefore, the higher level of h2-calponin in the basal layer of skin (Fig. 1) may reflect such a regulatory role during epidermal keratinocyte differentiation (67).
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The Role of h2-calponin in the Function of Actin Cytoskeleton—h2-calponin association to the actin filaments and its regulation by the mechanical tension of the cytoskeleton indicate functional significance. A previous study observed that transfective expression of green fluorescent protein fusion h1-calponin produced a higher resistance to cytochalasin B than that by transfection of green fluorescent protein fusion h2-calponin in NIH 3T3 fibroblast cells (12). Based on the assumption that 3T3 cells had no endogenous h2-calponion, untransfected 3T3 control was not included in that study for comparison (12). However, we have found that NIH 3T3 cells express a significant level of h2-calponin (Fig. 9A). In the presence of endogenous h2-calponin, the cells overexpressing green fluorescent protein fusion h2-calponin retained most stress fibers after treatment with 2.5 µM (1.2 µg/ml) cytochalasin B for 15 min (see Fig. 5 in Ref. 12). This actually reflects a significant resistance, and therefore, the previous results may indicate that both h1 and h2 calponins could confer a resistance to cytochalasin B treatment (Fig. 8). The notion that h1 and h2 calponins may have conserved function is consistent with another previous study that transfective expression of h1-calponin had effects on the proliferation rate of NIH 3T3 cells (71) similar to the results of forced expression of h2-calponin in SM3 cells (13).
Tropomyosin is a major component in the actin cytoskeleton (43) and a protein that binds calponin (1, 20). The co-localization of h2-calponin and tropomyosin in the actin stress fibers (Fig. 7) and the positive correlation between the levels of force-expressed h2-calponin and endogenous tropomyosin in SM3 cells (Fig. 8C) suggest a functional relationship. Tropomyosin has been shown to play a role in actin filament stability (42). Therefore, the effect of h2-calponin in stabilizing actin stress fibers as shown by resistance to cytochalasin B treatment (Fig. 8A) may be through enhancing the function of Tm. Like the effect of C-terminal fragment of caldesmon on actin filament stability (59), the relationship between h2-calponin and Tm in the structure and function of non-muscle cytoskeleton deserves further investigation. The finding that mechanical tension regulates the expression of h2-calponin with potential functional significance lays a foundation for understanding the physiological function of calponin.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Molecular Cardiology, Evanston Northwestern Healthcare, Evanston, IL 60201. Tel.: 847-570-1960; Fax: 847-570-1865; E-mail: jpjin{at}northwestern.edu.
2 The abbreviations used are: CMV, cytomegalovirus; PBS, phosphate-buffered saline; D-PBS, Dulbecco's PBS; mAb, monoclonal antibody; TRITC, tetramethylrhodamine isothiocyanate; Tm, tropomyosin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONREFERENCES |
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