Transforming Growth Factor-S Stimulates the Expression of Desmosomal Proteins in Bronchial Epithelial Cells Masami Yoshida, Debra J. Romberger, Mary G. Illig, Hajime Takizawa, Oliviero Sacco, John R. Spurzem, Joseph H. Sisson, Stephen I. Rennard, and Joe D. Beckmann University of Nebraska Medical Center, Department of Internal Medicine, Pulmonary and Critical Care Medicine Section, Omaha, Nebraska
Transforming growth factor-S, (TGF-,Bl) has been shown to induce squamous differentiation of cultured airway epithelial cells. It has also been shown to increase expression of matrix proteins and integrin receptors in cell culture of these and other cells. However, it is unknown if TGF-{3] affects expression of genes encoding intercellular junctional proteins. Therefore, we have investigated the effect of TGF-{3] on the expression of proteins and mRNAs for desmoplakins (DPs) I and II, desmosomal plaque proteins. Fibronectin, known to be induced by TGF-,Bl was used as a positive control and tubulin as a negative control. Twenty-four hours after TGF-,Bl stimulation, DP I and II mRNA levels assessed by Northern blotting analysis had increased significantly (DP I mRNA, 1.8-fold, P < 0.05; DP II mRNA, 2.4-fold, P < 0.04), thereby indicating pretranslational regulation of DP expression. By comparison, mRNA for fibronectin increased 8.1-foldwhereas mRNA for tubulin was unchanged. Immunofluorescence using the monoclonal anti-DP I and II antibodies revealed dramatic increased expression of punctate DP structures after exposure to TGF-{3]. Immunoblot analyses with polyclonal anti-DP I antibodies showed increased levels of both DP 1(250 kD) and DP II (215 kD), with the DP I increase being more pronounced (DP I, 2.5-fold; DP II, l.4-fold at 48 h relative to controls), suggesting translational regulation by TGF-{3]. This study therefore demonstrates the ability of TGF-,Bl to alter cellular phenotype by altering expression of proteins involved in intercellular junctions. Alteration of the expression of such proteins may be an important feature of TGF-,BJ-induced cellular differentiation.
Transforming growth factors-d (TGFs-{1) are a family of multifunctional polypeptides that are expressed and released by many cell types (1). These proteins are biologically active as disulfide-linked dimers (25 kD) that are derived from a much larger precursor polypeptide. One striking effect of TGF-{11 is its ability to induce squamous differentiation in many cell types, e.g., bronchial epithelial cells (2), endothelial cells (3), keratinocytes (4), and intestinal epithelial cells (5). In addition, TGFs-,B likely play an important role during wound healing in many cell types as described previ-
ously (6). Recent studies have made it clear that TGF-{31 induces the synthesis of several extracellular matrix proteins, including types I and III collagen and fibronectin (FN) (7-9). Furthermore, TGF-{31 is reported to increase the expression of (Received in original form July 17,1991 and in revised form September 19,
1991) Address correspondence to: Masami Yoshida, M.D., and Joe D. Beckmann, Ph.D., Department of Internal Medicine, Pulmonary and Critical Care Medicine Section, 600 South 42nd Street, Omaha, NE 68198-2465. Abbreviations: bovine bronchial epithelial cell, BBEC; desmoplakin, DP; enzyme-linked immunosorbent assay, ELISA; fibronectin, FN; sodium dodecyl sulfate, SDS; transforming growth factor-S, TGF-I3. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 439-445, 1992
cell surface receptors for these matrix proteins (1, 9). It is reasonable to postulate, therefore, that TGF-{1 might also affect intercellular attachment proteins. Desmosomes are intercellular adhesive junctions that are an important component in, the cohesion of epithelial cells. The cytoplasmic side of the assembled desmosomes is joined to tonofilaments, which are composed of cytokeratins. Therefore, desmosomes provide a link between the intermediate filament systems of adjacent epithelial cells. These junctional complexes have several components including the 250- and 215-kD desmoplakin (DP) proteins, which compose the dense intracellular plaque of the complex (10). In this article, we report that TGF-{11 increases the content of DPs I and II in cultured bovine bronchial epithelial cells (BBECs) concurrent with an increase in mRNAs encoding these proteins.
Materials and Methods Preparation of BBECs Primary BBECs were obtained by a modification (11, 12) of the method of Lee and colleagues (13). Bronchial basal cells were isolated by centrifugal fractionation (12) and cultured for two 1:4 passages in LHC-9/RPMI 1640 (1:1) (14) before plating on Vitrogen (Collagen Corp., Palo Alto, CA) coated tissue culture dishes at 37° C, with an atmosphere of 5 %
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CO 2 and 95% air (100-mm diameters for the extraction of protein and RNA, 35-mm diameters for the cell counts, and quantification of FN in the supernatant medium). Antibodies and cDNA A rabbit antiserum directed against the DP 1(250 kD) of bovine (tongue) desmosomes (15) and DP I and II cDNA (clone p5a) (16) were kindly provided by Drs. Jonathan C. R. Jones and Kathleen 1. Green (Northwestern University Medical School, Chicago, IL). Mouse monoclonal anti-bovine DP I and II antibody (Clone DP 215kD; ICN ImmunoBiologicals, Lisle, IL) (17) was used in these studies. Rabbit anti-bovine plasma FN antiserum prepared in our laboratory (11) was also used in enzyme-linked immunosorbent assay (ELISA). Fluorescein-conjugated rabbit anti-mouse IgG was purchased from Sigma Chemical Co. (St. Louis, MO). Human FN cDNA was a kind gift of Dr. Francisco E. Baralle (University of Oxford, Oxford, UK) (18). The tubulin cDNA clone, mouse m{35, was a kind gift from Dr. Don W. Cleveland (Johns Hopkins University, Baltimore, MD) (19, 20). Cell Number and Volume Cell numbers were calculated by a standard hemocytometer after washing and trypsinization. Cell volumes were measured as relative Coulter volume signals by the FACS Analyzer (Becton-Dickinson FACS Systems, San Jose, CA) interfaced with a Hewlett-Packard Computer, using log channel No. 0-250. The mean log channel volume numbers are linearly related to the cell volumes (21). Production of FN by BBECs Measured by ELISA Cells cultured to 70 to 75 % confluence were exposed to TGF-{3) in fresh media, and conditioned media were harvested at 0, 2, 6, 12, 24, and 48 h. FN levels in the conditioned media were measured by ELISA (22), using purified bovine plasma FN as standard. These were then divided by cell counts at the time of harvest and by the time elapsed since medium change in order to express FN production rate per cell. Immunolocalization BBECs were cultured on Lab-Tek 8 chamber slides (Miles Laboratories, Naperville, IL) coated with 33 Itg/ml Vitrogen. After removal of culture medium, the cells were fixed for 2 to 3 min in acetone (-20° C) and then air-dried. To minimize nonspecific binding of antibody, the fixed material was preadsorbed with normal goat serum diluted 1:20 in phosphate-buffered saline for 20 min at 37° C. Such preparations were then processed for single indirect immunofluorescence as previously reported (15), with monoclonal antibovine DP I and II antibody diluted 1:50 and polyclonal anti-human transferrin receptor antibody (Sigma) diluted 1:50 as negative control. Immunoblotting Procedure Whole BBEC proteins were solubilized with 8 M urea in 0.19 M Tris-HCI (pH 6.8), 1% sodium dodecyl sulfate (SDS), 1% {3-mercaptoethanol added to the culture dishes (23). After rocking at room temperature for 30 min, the solution was transferred into a 1.5-ml microcentrifuge tube and
vortexed for 6 min to shear DNA. After trichloroacetic acid precipitation steps, protein was then estimated by a modification (24) of the Lowry method (25), using bovine serum albumin as the standard. SDS gel electrophoresis using 5.0% acrylamide resolving gel with 4.0% stacking gel (26) was performed on 50 Itg of whole BBEC protein extracts. Duplicate gels were either stained with Coomassie R-250 or transferred to Immobilon Transfer Membrane (Millipore Corp., Bedford, MA) (27). Markers on the blots were identified by staining of the bound proteins with Ponceau S (Sigma). Immunodetection was done according to Johnson and colleagues (28). Densitometric analyses were performed after image transfer to Kodak 5561 Translite film (Eastman Kodak Co., Rochester, NY). RNA Preparation and Northern Blot Hybridization Total RNA of BBECs prepared as described previously (29) was washed with 80 % ethanol and solubilized in 0.5 % SDS at 65° C for 10 min. Before using for electrophoresis, total RNAs were quantitated by spectrophotometry (DU Series 62 Spectrophotometer; Beckman Instruments, Fullerton, CA). Total RNA samples denatured in formaldehyde/formamide (10 ltg/lane, containing 10 Itg ethidium bromide/sample) were electrophoretically separated on 0.8% agarose/2.2 M formaldehyde gels (30) and directly transferred to nitrocellulose membranes (S&S NC BA 85 45 Itm; Scheicher & Schuell, Keene, NH) by capillary action using 20 X SSPE. cDNA probes, which were excised from plasmid vectors and purified by agarose gel electrophoresis, were radiolabeled by random hexamer priming as described previously (31). Blots were probed with 32P-Iabeled cDNAs under standard conditions as described (32). Final washings were carried out at 42° C in O.1x SSPE/O.1 % SDS for 1 h. Exposures to Kodak X-AR film were made at -70° C with one intensifying screen. Hybridization signals were quantified by densitometric scanning with a GS 300 Transmittance/Reflectance Scanning Densitometer (Hoefer Scientific Instruments, San Francisco, CA). Statistics Student's t test was used to make all comparisons.
Results Effect of TGF-{3t on Cell Morphology and Cell Volume Other investigators have reported that TGF-{3] induces squamous differentiation of airway epithelial cells. Therefore, we initially examined the effect of TGF-{3] on BBEC morphology. BBECs exposed to 200 pM TGF-{3] changed from a "cobblestone" morphology (Figure 1a) to large, flattened cells containing single nuclei (Figure 1b), similar to the squamous phenotype described previously (2). The change of the cell morphology was accompanied by a change in cell volume: for cells exposed to 200 pM TGF-{3I, the relative volume as determined by log mean channel increased from . 170.15 ± 3.86 (mean ± SEM) to 199.40 ± 10.80 after 48 h exposure to 200 pM TGF-{3] (P = 0.044, n = 4). This change indicates only a 17% average increase of cell volume. Therefore, the overt morphologic changes observed by phase microscopy are not coupled to large volumetric changes in response to TGF-{3].
Yoshida, Romberger, Illig et al.: lGF-13 Stimulates Desmoplakin Gene Expression
441
Figure 1. Appearance of bovine bronchial epithelial cells (BBECs) incubated in the absence or presence of transforming growth factor-{3, (TGF-13,) by phase-contrast microscopy. BBEC cultures in LHC9 mixed with RPMI 1640 (1:1) on Lab-Tek 8 chamber slides coated with 33 /Lg/ml Vitrogen were incubated in the absence (panel a) or presence (panel b) of 200 pM TGF-I3, for 48 h. Bars = 100 J-tm .
Immunofluorescence Observation The localization and distribution of DP I and II in BBECs were determined by indirect immunofluorescence, using the monoclonal anti-DP antibody (33). In control cells (Figure 2a), immunofluorescence staining was of moderate intensity and diffusely spread throughout the cytoplasm. Punctate staining arrays along a part of the cell boundries were also seen. Forty-eight hours after addition of lGF-I3, (Figure 2b) , the cytoplasmic fluorescence was reduced and characteristic punctate staining of DP antibody binding sites along cell boundaries, corresponding to cell-to-cell contact regions, increased in intensity in monolayer cultures. In contrast, no such increase in staining occurred when BBECs treated with
Figure 2. Immunofluorescent staining of BBECmonolayer cultures with monoclonal antibovinedesmoplakin (DP) I and II antibodies (panels a and b) and polyclonal anti-humantransferrin receptor antibody (panel c, negative control). Panel a: In nonTGF-I3,-treated cells, immunofluorescence was of moderate intensity and diffusely spread throughout the cytoplasm. Punctate stainingarraysalonga part of the cell boundaries were also seen. Panel b: In cells treated with 200 pM TGF-{3, for 48 h, desmosomal staining at cell periphery increased in intensity, and cytoplasmic fluorescence was reduced. Panel c: Negative control: cells were treated with 200 pM TGF-{3, for 48 h. The background on this immunofluorescent staining is shown. Bars = 10 J-tm.
200 pM TGF-I3, for 48 h were stained with poly clonal anti -human transferrin receptor antibody (Figure 2c) . Immunoblot Analyses of Whole BBEC Protein The qualitative results derived from immunofluorescence have been extended by immunoblot analyses. This approach allows a comparison between the 250-kD (DP I) and 2l5-kD (DP II) protein levels. The DP antiserum reacted with several proteins extracted from cultured BBECs (Figure 3a) except hemidesmosomal components (34). DP I staining intensity increased with time after exposure to 200 pM lGF-I3 , (96% increase at 48 h as determined by densitometry; see MATERIALS AND METHODS). Changes of DP II levels (22 %
442
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992
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Figure 3. Effect of TGF-131 on DP protein expression by cultured BBECs . Total proteins were extracted using sodium dodecyl sulfate (SDS)/urea (see MATERIALS AND METHODS) followed by SDS-polyacrylamide gel electrophoresis and irnmunoblot analysis. Each lane contained 50 J.l.g protein. Panel s a and b illustrate the time course for the response to 200 pM TGF-131 exposure. Panels c and d show the response to varied doses of TGF-131 for 48 h. Panels a and c: Blots probed with anti-DP antibody (see MATERIALS AND METHODS). Panels band d: Replicate gels stained with Coomassie R-250 .
increase at 48 h) were not as prominent as the increase in DP I. The lower molecular weight immunoreactive peptides, probably degradation products of DPs (35, 36), decreased after exposure to TGF-I3I' in contrast to the increases of the DP I band . This result suggests that TGF-131 may act at a post-translational level to inhibit the degradation of DP I and II. Although proteolysis may be an artifact of cell solubilization, the uniformity of the stained gels (Figure 3b) does not reveal any overt sample degradation. The effect of varied doses of TGF-131for 48 h on the expression of DP I and II has also been determined (Figures 3c and 3d). Once again, DP I was most responsive to TGF-I3I' exhibiting a 135% increase with 200 pM TGF-13 in this experiment. A 43 % increase of DP I staining was observed with 10 pM TGF-I3I' Summation of all immunodetectable bands indicated a 60 to 70 % increase due to 100 to 300 pM TGF-I3I. As a positive marker of the effect of TGF-131on BBECs, we saved the supernatant media after addition of TGF-131 and measured FN in conditioned media by ELISA. BBECs were induced by TGF-131 to release significantly more FN into the culture media than control cells at 2 (P = 0.0461),
12 (P = 0.0259), 24 (P = 0 .0008), and 48 h (P = 0.0018) (Figure 4). This result confirms our previous observations (37) and provides a basis for comparison of the response of other genes to TGF-131 (see below). TGF-I3. Increases DP I and II mRNA Levels in BBECs Other investigators have reported that airway epithelial cells undergo spontaneous squamous differentiation upon reaching confluence (2). Therefore, it was first necessary to show the relationship between the expression of DP I .and II mRNA and the density of cultured BBECs . As we were interested in modulation of DP I and II mRNA , we elected to study a variety of subconfluent cell densities. Total RNAs extracted from BBECs cultures of approximately 40, 70, and 90% confluency were analyzed by Northern blot hybridization with a DP I and II cDNA probe. Specific hybridization of bands of 9.5 kb and 7.5 kb (16) corresponding to DP I and II mRNA were observed . The expressions of DP I and II mRNA increased slightly with culture confluence (Figure 5); however, stimulation with TGF-131 for 24 h increased DP I and II mRNA levels above those observed in control cultures . Therefore, subsequent experiments are not overtly
Yoshida, Romberger, Illig et al. : TGF-13 Stimulates Desmoplakin Gene Expression
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Transforming growth factor-S, (TGF-,Bl) has been shown to induce squamous differentiation of cultured airway epithelial cells. It has also been shown to increase expression of matrix proteins and integrin receptors in cell culture of these and other cells. However, it is unknown if TGF-{3] affects expression of genes encoding intercellular junctional proteins. Therefore, we have investigated the effect of TGF-{3] on the expression of proteins and mRNAs for desmoplakins (DPs) I and II, desmosomal plaque proteins. Fibronectin, known to be induced by TGF-,Bl was used as a positive control and tubulin as a negative control. Twenty-four hours after TGF-,Bl stimulation, DP I and II mRNA levels assessed by Northern blotting analysis had increased significantly (DP I mRNA, 1.8-fold, P < 0.05; DP II mRNA, 2.4-fold, P < 0.04), thereby indicating pretranslational regulation of DP expression. By comparison, mRNA for fibronectin increased 8.1-foldwhereas mRNA for tubulin was unchanged. Immunofluorescence using the monoclonal anti-DP I and II antibodies revealed dramatic increased expression of punctate DP structures after exposure to TGF-{3]. Immunoblot analyses with polyclonal anti-DP I antibodies showed increased levels of both DP 1(250 kD) and DP II (215 kD), with the DP I increase being more pronounced (DP I, 2.5-fold; DP II, l.4-fold at 48 h relative to controls), suggesting translational regulation by TGF-{3]. This study therefore demonstrates the ability of TGF-,Bl to alter cellular phenotype by altering expression of proteins involved in intercellular junctions. Alteration of the expression of such proteins may be an important feature of TGF-,BJ-induced cellular differentiation.
Transforming growth factors-d (TGFs-{1) are a family of multifunctional polypeptides that are expressed and released by many cell types (1). These proteins are biologically active as disulfide-linked dimers (25 kD) that are derived from a much larger precursor polypeptide. One striking effect of TGF-{11 is its ability to induce squamous differentiation in many cell types, e.g., bronchial epithelial cells (2), endothelial cells (3), keratinocytes (4), and intestinal epithelial cells (5). In addition, TGFs-,B likely play an important role during wound healing in many cell types as described previ-
ously (6). Recent studies have made it clear that TGF-{31 induces the synthesis of several extracellular matrix proteins, including types I and III collagen and fibronectin (FN) (7-9). Furthermore, TGF-{31 is reported to increase the expression of (Received in original form July 17,1991 and in revised form September 19,
1991) Address correspondence to: Masami Yoshida, M.D., and Joe D. Beckmann, Ph.D., Department of Internal Medicine, Pulmonary and Critical Care Medicine Section, 600 South 42nd Street, Omaha, NE 68198-2465. Abbreviations: bovine bronchial epithelial cell, BBEC; desmoplakin, DP; enzyme-linked immunosorbent assay, ELISA; fibronectin, FN; sodium dodecyl sulfate, SDS; transforming growth factor-S, TGF-I3. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 439-445, 1992
cell surface receptors for these matrix proteins (1, 9). It is reasonable to postulate, therefore, that TGF-{1 might also affect intercellular attachment proteins. Desmosomes are intercellular adhesive junctions that are an important component in, the cohesion of epithelial cells. The cytoplasmic side of the assembled desmosomes is joined to tonofilaments, which are composed of cytokeratins. Therefore, desmosomes provide a link between the intermediate filament systems of adjacent epithelial cells. These junctional complexes have several components including the 250- and 215-kD desmoplakin (DP) proteins, which compose the dense intracellular plaque of the complex (10). In this article, we report that TGF-{11 increases the content of DPs I and II in cultured bovine bronchial epithelial cells (BBECs) concurrent with an increase in mRNAs encoding these proteins.
Materials and Methods Preparation of BBECs Primary BBECs were obtained by a modification (11, 12) of the method of Lee and colleagues (13). Bronchial basal cells were isolated by centrifugal fractionation (12) and cultured for two 1:4 passages in LHC-9/RPMI 1640 (1:1) (14) before plating on Vitrogen (Collagen Corp., Palo Alto, CA) coated tissue culture dishes at 37° C, with an atmosphere of 5 %
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992
CO 2 and 95% air (100-mm diameters for the extraction of protein and RNA, 35-mm diameters for the cell counts, and quantification of FN in the supernatant medium). Antibodies and cDNA A rabbit antiserum directed against the DP 1(250 kD) of bovine (tongue) desmosomes (15) and DP I and II cDNA (clone p5a) (16) were kindly provided by Drs. Jonathan C. R. Jones and Kathleen 1. Green (Northwestern University Medical School, Chicago, IL). Mouse monoclonal anti-bovine DP I and II antibody (Clone DP 215kD; ICN ImmunoBiologicals, Lisle, IL) (17) was used in these studies. Rabbit anti-bovine plasma FN antiserum prepared in our laboratory (11) was also used in enzyme-linked immunosorbent assay (ELISA). Fluorescein-conjugated rabbit anti-mouse IgG was purchased from Sigma Chemical Co. (St. Louis, MO). Human FN cDNA was a kind gift of Dr. Francisco E. Baralle (University of Oxford, Oxford, UK) (18). The tubulin cDNA clone, mouse m{35, was a kind gift from Dr. Don W. Cleveland (Johns Hopkins University, Baltimore, MD) (19, 20). Cell Number and Volume Cell numbers were calculated by a standard hemocytometer after washing and trypsinization. Cell volumes were measured as relative Coulter volume signals by the FACS Analyzer (Becton-Dickinson FACS Systems, San Jose, CA) interfaced with a Hewlett-Packard Computer, using log channel No. 0-250. The mean log channel volume numbers are linearly related to the cell volumes (21). Production of FN by BBECs Measured by ELISA Cells cultured to 70 to 75 % confluence were exposed to TGF-{3) in fresh media, and conditioned media were harvested at 0, 2, 6, 12, 24, and 48 h. FN levels in the conditioned media were measured by ELISA (22), using purified bovine plasma FN as standard. These were then divided by cell counts at the time of harvest and by the time elapsed since medium change in order to express FN production rate per cell. Immunolocalization BBECs were cultured on Lab-Tek 8 chamber slides (Miles Laboratories, Naperville, IL) coated with 33 Itg/ml Vitrogen. After removal of culture medium, the cells were fixed for 2 to 3 min in acetone (-20° C) and then air-dried. To minimize nonspecific binding of antibody, the fixed material was preadsorbed with normal goat serum diluted 1:20 in phosphate-buffered saline for 20 min at 37° C. Such preparations were then processed for single indirect immunofluorescence as previously reported (15), with monoclonal antibovine DP I and II antibody diluted 1:50 and polyclonal anti-human transferrin receptor antibody (Sigma) diluted 1:50 as negative control. Immunoblotting Procedure Whole BBEC proteins were solubilized with 8 M urea in 0.19 M Tris-HCI (pH 6.8), 1% sodium dodecyl sulfate (SDS), 1% {3-mercaptoethanol added to the culture dishes (23). After rocking at room temperature for 30 min, the solution was transferred into a 1.5-ml microcentrifuge tube and
vortexed for 6 min to shear DNA. After trichloroacetic acid precipitation steps, protein was then estimated by a modification (24) of the Lowry method (25), using bovine serum albumin as the standard. SDS gel electrophoresis using 5.0% acrylamide resolving gel with 4.0% stacking gel (26) was performed on 50 Itg of whole BBEC protein extracts. Duplicate gels were either stained with Coomassie R-250 or transferred to Immobilon Transfer Membrane (Millipore Corp., Bedford, MA) (27). Markers on the blots were identified by staining of the bound proteins with Ponceau S (Sigma). Immunodetection was done according to Johnson and colleagues (28). Densitometric analyses were performed after image transfer to Kodak 5561 Translite film (Eastman Kodak Co., Rochester, NY). RNA Preparation and Northern Blot Hybridization Total RNA of BBECs prepared as described previously (29) was washed with 80 % ethanol and solubilized in 0.5 % SDS at 65° C for 10 min. Before using for electrophoresis, total RNAs were quantitated by spectrophotometry (DU Series 62 Spectrophotometer; Beckman Instruments, Fullerton, CA). Total RNA samples denatured in formaldehyde/formamide (10 ltg/lane, containing 10 Itg ethidium bromide/sample) were electrophoretically separated on 0.8% agarose/2.2 M formaldehyde gels (30) and directly transferred to nitrocellulose membranes (S&S NC BA 85 45 Itm; Scheicher & Schuell, Keene, NH) by capillary action using 20 X SSPE. cDNA probes, which were excised from plasmid vectors and purified by agarose gel electrophoresis, were radiolabeled by random hexamer priming as described previously (31). Blots were probed with 32P-Iabeled cDNAs under standard conditions as described (32). Final washings were carried out at 42° C in O.1x SSPE/O.1 % SDS for 1 h. Exposures to Kodak X-AR film were made at -70° C with one intensifying screen. Hybridization signals were quantified by densitometric scanning with a GS 300 Transmittance/Reflectance Scanning Densitometer (Hoefer Scientific Instruments, San Francisco, CA). Statistics Student's t test was used to make all comparisons.
Results Effect of TGF-{3t on Cell Morphology and Cell Volume Other investigators have reported that TGF-{3] induces squamous differentiation of airway epithelial cells. Therefore, we initially examined the effect of TGF-{3] on BBEC morphology. BBECs exposed to 200 pM TGF-{3] changed from a "cobblestone" morphology (Figure 1a) to large, flattened cells containing single nuclei (Figure 1b), similar to the squamous phenotype described previously (2). The change of the cell morphology was accompanied by a change in cell volume: for cells exposed to 200 pM TGF-{3I, the relative volume as determined by log mean channel increased from . 170.15 ± 3.86 (mean ± SEM) to 199.40 ± 10.80 after 48 h exposure to 200 pM TGF-{3] (P = 0.044, n = 4). This change indicates only a 17% average increase of cell volume. Therefore, the overt morphologic changes observed by phase microscopy are not coupled to large volumetric changes in response to TGF-{3].
Yoshida, Romberger, Illig et al.: lGF-13 Stimulates Desmoplakin Gene Expression
441
Figure 1. Appearance of bovine bronchial epithelial cells (BBECs) incubated in the absence or presence of transforming growth factor-{3, (TGF-13,) by phase-contrast microscopy. BBEC cultures in LHC9 mixed with RPMI 1640 (1:1) on Lab-Tek 8 chamber slides coated with 33 /Lg/ml Vitrogen were incubated in the absence (panel a) or presence (panel b) of 200 pM TGF-I3, for 48 h. Bars = 100 J-tm .
Immunofluorescence Observation The localization and distribution of DP I and II in BBECs were determined by indirect immunofluorescence, using the monoclonal anti-DP antibody (33). In control cells (Figure 2a), immunofluorescence staining was of moderate intensity and diffusely spread throughout the cytoplasm. Punctate staining arrays along a part of the cell boundries were also seen. Forty-eight hours after addition of lGF-I3, (Figure 2b) , the cytoplasmic fluorescence was reduced and characteristic punctate staining of DP antibody binding sites along cell boundaries, corresponding to cell-to-cell contact regions, increased in intensity in monolayer cultures. In contrast, no such increase in staining occurred when BBECs treated with
Figure 2. Immunofluorescent staining of BBECmonolayer cultures with monoclonal antibovinedesmoplakin (DP) I and II antibodies (panels a and b) and polyclonal anti-humantransferrin receptor antibody (panel c, negative control). Panel a: In nonTGF-I3,-treated cells, immunofluorescence was of moderate intensity and diffusely spread throughout the cytoplasm. Punctate stainingarraysalonga part of the cell boundaries were also seen. Panel b: In cells treated with 200 pM TGF-{3, for 48 h, desmosomal staining at cell periphery increased in intensity, and cytoplasmic fluorescence was reduced. Panel c: Negative control: cells were treated with 200 pM TGF-{3, for 48 h. The background on this immunofluorescent staining is shown. Bars = 10 J-tm.
200 pM TGF-I3, for 48 h were stained with poly clonal anti -human transferrin receptor antibody (Figure 2c) . Immunoblot Analyses of Whole BBEC Protein The qualitative results derived from immunofluorescence have been extended by immunoblot analyses. This approach allows a comparison between the 250-kD (DP I) and 2l5-kD (DP II) protein levels. The DP antiserum reacted with several proteins extracted from cultured BBECs (Figure 3a) except hemidesmosomal components (34). DP I staining intensity increased with time after exposure to 200 pM lGF-I3 , (96% increase at 48 h as determined by densitometry; see MATERIALS AND METHODS). Changes of DP II levels (22 %
442
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992
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Figure 3. Effect of TGF-131 on DP protein expression by cultured BBECs . Total proteins were extracted using sodium dodecyl sulfate (SDS)/urea (see MATERIALS AND METHODS) followed by SDS-polyacrylamide gel electrophoresis and irnmunoblot analysis. Each lane contained 50 J.l.g protein. Panel s a and b illustrate the time course for the response to 200 pM TGF-131 exposure. Panels c and d show the response to varied doses of TGF-131 for 48 h. Panels a and c: Blots probed with anti-DP antibody (see MATERIALS AND METHODS). Panels band d: Replicate gels stained with Coomassie R-250 .
increase at 48 h) were not as prominent as the increase in DP I. The lower molecular weight immunoreactive peptides, probably degradation products of DPs (35, 36), decreased after exposure to TGF-I3I' in contrast to the increases of the DP I band . This result suggests that TGF-131 may act at a post-translational level to inhibit the degradation of DP I and II. Although proteolysis may be an artifact of cell solubilization, the uniformity of the stained gels (Figure 3b) does not reveal any overt sample degradation. The effect of varied doses of TGF-131for 48 h on the expression of DP I and II has also been determined (Figures 3c and 3d). Once again, DP I was most responsive to TGF-I3I' exhibiting a 135% increase with 200 pM TGF-13 in this experiment. A 43 % increase of DP I staining was observed with 10 pM TGF-I3I' Summation of all immunodetectable bands indicated a 60 to 70 % increase due to 100 to 300 pM TGF-I3I. As a positive marker of the effect of TGF-131on BBECs, we saved the supernatant media after addition of TGF-131 and measured FN in conditioned media by ELISA. BBECs were induced by TGF-131 to release significantly more FN into the culture media than control cells at 2 (P = 0.0461),
12 (P = 0.0259), 24 (P = 0 .0008), and 48 h (P = 0.0018) (Figure 4). This result confirms our previous observations (37) and provides a basis for comparison of the response of other genes to TGF-131 (see below). TGF-I3. Increases DP I and II mRNA Levels in BBECs Other investigators have reported that airway epithelial cells undergo spontaneous squamous differentiation upon reaching confluence (2). Therefore, it was first necessary to show the relationship between the expression of DP I .and II mRNA and the density of cultured BBECs . As we were interested in modulation of DP I and II mRNA , we elected to study a variety of subconfluent cell densities. Total RNAs extracted from BBECs cultures of approximately 40, 70, and 90% confluency were analyzed by Northern blot hybridization with a DP I and II cDNA probe. Specific hybridization of bands of 9.5 kb and 7.5 kb (16) corresponding to DP I and II mRNA were observed . The expressions of DP I and II mRNA increased slightly with culture confluence (Figure 5); however, stimulation with TGF-131 for 24 h increased DP I and II mRNA levels above those observed in control cultures . Therefore, subsequent experiments are not overtly
Yoshida, Romberger, Illig et al. : TGF-13 Stimulates Desmoplakin Gene Expression
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