—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 E. 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 (ELISA) and subcloned three times to establish stable cell line. The mAbs were produced in the forms of hybridoma cultural supernatant and mouse ascites fluids. ELISA Immunoglobulin isotyping (Invitrogen) determined its subclass as IgG2b κ. 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, CGβ6 against hTM2 and hTM3, LC24 against hTM4 and CG3 against hTM5, were described previously (40
). A rabbit anti-human involucrin antibody was produced as previously described (44
—SDS-polyacrylamide gel electrophoresis (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 nitroblue tetrazolium chromogenic substrate reaction. Purified h2- and h1-calponins (37
) was used as control.
—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 has no cross-reaction to h1-calponin, followed by horseradish peroxidase-labeled anti-mouse IgG second antibody (Sigma) and H2
-daminobenzidin substrate reaction using standard immunohistochemical method (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 sec.
—Primary keratinocytes were isolated from human foreskin samples (47
) and cultured in keratinocyte serum-free media (KSFM, 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
Human fibroblast cell line KD (American Type Culture Collection, CRL 1295) was cultured in DMEM (Dulbecco’s modified Eagle medium) containing 10% fetal bovine serum (FBS), penicillin (100 i.u./ml) and streptomycin (50 i.u./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 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 DMEM containing 10% FBS, penicillin (100 i.u./ml), streptomycin (50 i.u./ml), and G418 (100 μg/ml) at 37°C in 5% CO2
—Precleaned glass cover slips were coated with 0.1% gelatin and dried under UV radiation for 3 hours before placed in the culture dish. Keratinocytes and KD cells were seeded to grow monolayer on the cover slips. 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 Na2
, 1.47 mM KH2
, pH 7.2). The cells were then fixed with cold acetone for 20 minutes. 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. Tetramethylrhodamine isothiocyanate (TRITC)- or fluorescein isothiocyanate (FITC)-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, pre-confluent keratinocytes were trypsinized and seeded at high and low densities (initial density = 9 × 105 cells and 3 ×105 cells per 60 mm dish, respectively). The cells were suspended from the culture dishes after 1, 2, 3, 4 and 5 days using the Versene solution (in mM: 0.537 EDTA, 136.8 NaCl, 2.68 KCl, 8.1 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 lyze 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 ibration 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 lyzed 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 cover slips to provide soft matrix for cell culture as described previously (30
, 35, 52
). The cover slips were passed briefly through the inner flame of a Bunsen burner. A drop of 0.1N NaOH was smeared across the surface of the cover slip and air-dried. Thereafter, the cover slips 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; and soft, 3% gel with an acrylamide:bisacrylamide ratio of 74:1) were polymerized in between the treated cover slip at the bottom and an untreated cover slip on the top. After polymerization, the untreated cover slip 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 bis
-acrylamide cross-linker (30
After treating the gel with a bifunctional cross-linker (1 mM sulfo-SANPAH, Pierce, Rockford, IL) in 50 mM HEPES, pH 8.5 (30
), Type I collagen was applied to the gel surface, followed by gently shaking overnight at 4°C for cross linking to the gel. Gels were then washed with D-PBS and sterilized with UV irradiation for 3 hours. The gel was soaked in culture media at 37 °C for 1 hour before plating cells.
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 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 2, 4, 8, 12, 24, 48, and 72 hrs 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 hours 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
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 cover slips. After 3 days of culture, the cells were treated with 0.5, 0.75 and 1.0μg/ml of cytochalasin B (Sigma) at 37 °C for 30 min. h2-calponin-negative SM3 cells were examined as control. The cytochalasin B stock was prepared in DMSO, so a 0.2 % DMSO-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.
—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 cover slips (3 × 104
cells/35 mm dish). After 3 days of monolayer culture, the cells were treated with 100 μM blebbistatin, a myosin II ATPase inhibitor (56
), for 3 days. Parallel cultures in normal media or containing 0.2 % DMSO (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 cover slips 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 dpi 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 cell area, h2-calponin and tropomyosin levels are presented as mean ± SD. Statistical analysis was done using the Microsoft Excel computer program.