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Epithelial cell layers exhibit an ordered polarized architecture. However, such structures are disrupted during malignant transformation, which generally coincides with a loss of regulate cell growth. We are investigating how the cadherin cell adhesion system controls these processes. Cadherins form a molecular complex with alpha-catenin, and beta-catenin or plakoglobin at the cytoplasmic side in normal cells. Lung carcinoma PC9 cells express E-cadherin. Although they express other catenins, they lack alpha-catenin and cannot firmly aggregate, suggesting that their E-cadherin is inactive. Transfection of the PC9 cells with alpha-catenin cDNA leads to activation of the E-cadherin, inducing their compact aggregation. In these aggregates, an almost complete epithelial-specific architecture is organized, including the formation of microvilli and a junctional complex. We also studied the effect of hepatocyte growth factor/scatter factor (HGF/SF) on cell-cell contacts in keratinocyte cell lines, and found that this growth factor can disrupt desmosomal cell-cell contacts. HGF/SF, and also epidermal growth factor, enhance tyrosine phosphorylation of beta-catenin or plakoglobin in human carcinoma lines as they induce scattering of these cells. These findings suggest that the cadherin adhesion system is central in organizing epithelial structures and that tyrosine phosphorylation of catenins may modulate this organization process.

Sequential changes of gap junctions (GJs), tight junctions (TJs) and desmosomes (DSs) between hepatocytes during restorative proliferation were studied in rats after a single intraperitoneal administration of 200 mg/kg thioacetamide (TAA). Antibody against connexin 32 was used to demonstrate GJs; simultaneously the changes in TJs and DSs were studied using antibodies against 7H6 protein and desmoplakins. Propidium iodide and bromodeoxyuridine were used to recognize necrotic and proliferative cells. GJs were evenly distributed in early necrotic hepatocytes at 16 h after TAA treatment, then disappeared from necrotic and surrounding cells at 24 h. At 48 h, GJs had disappeared completely from hepatocytes in whole liver lobules, while many hepatocytes were heavily labelled with BrdU. At 72 h, GJs reappeared, firstly in perinecrotic areas. At 96 h after treatment, when the injured areas had disappeared and restorative proliferation ceased, GJs were distributed evenly throughout the lobules. Immunohistochemical observation of GJs in centrilobular, perinecrotic and periportal areas after TAA-induced hepatic necrosis was confirmed by counting the number of connexin-32-positive spots in the respective areas. TJs and DSs disappeared from necrotic cells at 24 h, but then increased between 24 and 48 h in perinecrotic areas, though the increased intensity of these junctions was more evident at 48 h. At 72 h, localization of TJs and DSs returned to normal. These results suggest that during the course of acute hepatic injury, GJs (cell-cell communication) behave differently from other intercellular junctions.

To investigate the possible role(s) of cytoskeletal elements in desmosome assembly we have studied the effects of cytostatic drugs on the assembly of desmosomes in MDCK epithelial cells. We showed previously [Pasdar et al.: Cell Motil. Cytoskeleton 23:201-213, 1992] that selective disruption of microtubules has no effect on desmosome assembly. Here, we have treated MDCK cells with cytochalasin B and a combination of cytochalasin B and nocodazole and analysed the effects of desmosome assembly. Immunofluorescence analysis of MDCK cultures following drug treatment indicated complete disruption of actin microfilaments and disorganization of cytokeratin intermediate filaments. Biochemical analysis of newly synthesized desmosomal membrane core glycoproteins as well as the cell adhesion protein E-cadherin revealed no effect of these drugs on the kinetics of synthesis, intracellular processing, or transport to the plasma membrane either in the presence or absence of cell-cell contact. However, morphological analyses revealed a significant disruption in the spatial organization of desmosomal proteins and E-cadherin. Drug treatment in the absence of cell-cell contact resulted in the disruption of the normally observed homogeneous punctate staining pattern and appearance of aggregate staining. Induction of cell-cell contact in these cultures resulted in redistribution of some of the aggregate staining to the plasma membrane. In contrast to control cultures, significant amount of intracellular staining was retained for all desmosomal proteins. Biochemical analyses of turnover rates of newly synthesized desmosomal proteins indicated a significant decrease in metabolic stability of these proteins while the turnover rate of E-cadherin was not significantly different among control and drug-treated cultures. Taken together, these results suggest that intact actin and cytokeratin filaments are necessary for the stability, efficient assembly, and spatial organization of the junctional components at the membrane. The regulatory role of cytokeratins and actin filaments in assembly and stability of desmosomes on the plasma membrane is discussed.

The hemidesmosome is an adhesion structure of the epidermal-dermal junction in keratinocytes. When keratinocytes migrate laterally or upward to differentiate, they must control the formation and disintegration of the hemidesmosomes. When keratinocytes are cultured in low-calcium (below 0.1 mM) medium, all cells behave like basal cells, adhere to the culture dish, and proliferate without differentiation. The calcium addition induces the differentiation. A bullous pemphigoid antigen, 180-kDa BPA, has been shown to be a component of the hemidesmosome. Using a monoclonal antibody to the 180-kDa BPA and a human squamous cell carcinoma cell line (DJM-1 cells), the fate of hemidesmosomes was studied after the addition of calcium to low-calcium-grown cells and 12-tetradecanoylphorbol-13-acetate (TPA) to high-calcium (1.87 mM) grown cells by immunofluorescence and immunoelectron microscopy. The antigen was distributed evenly as fine dots on the entire ventral surface of low-calcium cells, whereas they formed a peculiar, concentric ring or arch arrangement on the ventral surface of high-calcium cells. Immunoelectron microscopy revealed the deposits of gold particles at sites on the membrane surface, where some filamentous or electron-dense materials were associated, although the complete structure of hemidesmosomes was not formed. They deposited directly onto the membrane surface in low-calcium cells and with a distance of 20-50 nm from the membrane surface in high-calcium cells. The calcium addition caused a profound reduction of the 180-kDa BPA-positive area for 30 to 120 min and then formed the high-calcium-ring pattern after 4 to 6 h. A similar calcium response was seen in normal human keratinocytes. TPA (16 nM) treatment caused disintegration of the ring pattern in high-calcium DJM-1 cells. This was inhibited with a protein kinase C (PKC) inhibitor. H7 (20 microM). These results suggest that the hemidesmosome is a dynamic structure and PKC can be one of the major factors in controlling the hemidesmosome, since it is known that the low-high calcium shift induces a calcium influx and a PKC activation, and TPA activates PKC in keratinocytes.

Short-chain monocarboxylic acids (MCAs) selectively protect desmosomal junctions of MDCK cells from disruption by chelating agents and low calcium medium. This effect occurs in the millimolar concentration range and increases inversely with carbon chain length (formate > acetate = propionate > butyrate > isobutyrate > isovalerate). The relative activity of MCAs does not correlate with their overall hydrophobicity or ability to chelate ions, or their effectiveness in lowering cytosolic pH. It exhibits chemical specificity and is dependent upon postconfluency culture age. MCAs also inhibit cell rounding produced by low concentrations of aminocarboxylate-chelating agents. Their effect on cell rounding, but not on desmosomes, can be antagonized by okadaic acid. The possibility is discussed that MCAs may produce their effects by binding specifically to protein(s) associated with the desmosome of mature, fully polarized MDCK monolayers.

Treatment of human epithelial cells in culture with phorbol esters (TPA) gives rise to a transient and reversible loss of accessibility to antibodies of the nonhelical carboxy-terminal domain of nuclear lamin A that distinguishes it from lamin C. No change in the accessibility of epitopes present in the common domain of lamins A and C was observed. Loss of accessibility of lamin A was not due to proteolytic degradation nor to modification of the isoelectric point of lamin A and did not depend upon protein kinase C activation nor protein synthesis. Perturbation of desmosome organization by growth in low calcium blocked the effect of TPA on lamin A. Prolonged exposure to nocodazole, one of the effects of which is a perinuclear collapse of intermediate filaments, also blocked the effect of TPA on lamin A. These results suggest that the initial target of TPA may be at the level of cell-cell contacts and that the perturbation induced by TPA may be propagated via the structural link formed by intermediate filaments between the cell surface and the nucleus, giving rise to a change in conformation of the carboxy-terminal domain of lamin A or to an interaction of this domain with another nuclear component. These results form the basis for the hypothesis that the interphase nuclear lamina may play an active role in the process of mechanochemical signal transduction.

Transforming growth factor-beta 1 (TGF-beta 1) 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-beta 1 affects expression of genes encoding intercellular junctional proteins. Therefore, we have investigated the effect of TGF-beta 1 on the expression of proteins and mRNAs for desmoplakins (DPs) I and II, desmosomal plaque proteins. Fibronectin, known to be induced by TGF-beta 1 was used as a positive control and tubulin as a negative control. Twenty-four hours after TGF-beta 1 stimulation, DP I and II mRNA levels assessed by Northern blotting analysis had increased significantly (DP I mRNA, 1.8-fold, P less than 0.05; DP II mRNA, 2.4-fold, P less than 0.04), thereby indicating pretranslational regulation of DP expression. By comparison, mRNA for fibronectin increased 8.1-fold whereas 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-beta 1. Immunoblot analyses with polyclonal anti-DP I antibodies showed increased levels of both DP I (250 kD) and DP II (215 kD), with the DP I increase being more pronounced (DP I, 2.5-fold; DP II, 1.4-fold at 48 h relative to controls), suggesting translational regulation by TGF-beta 1. This study therefore demonstrates the ability of TGF-beta 1 to alter cellular phenotype by altering expression of proteins involved in intercellular junctions.(ABSTRACT TRUNCATED AT 250 WORDS)

Using the rat bladder carcinoma cell line NBT-II we showed that collagens but not laminin and fibronectin were able to induce cell scattering. Acidic fibroblast growth factor and transforming growth factor alpha also promoted NBT-II cell dispersion on glass or tissue culture plastic. We have now further analysed the scatter response to these two growth factors in the presence of extracellular matrix molecules. In the presence of growth factors, no peripheral single-cell dispersion occurred on fibronectin and laminin, although time-lapse video analyses revealed intense cell mingling and motility inside the monolayer forming around NBT-II aggregates. Patterns of strings or files of cells protruding from the monolayer were often observed. The presence of a scattering activity in the complex acellular extracellular matrix deposited by NBT-II cells themselves strongly suggested that substratum conditioning was responsible for this effect. On the other hand, the two growth factors accelerated collagen-mediated NBT-II individual cell dispersion and locomotion in a reversible way. As a marker of cell dissociation, we studied desmosome distribution in aggregate cultures: desmosomes were present in aggregates formed in suspension even in the presence of growth factors, whereas internalization occurred after cell-to-substratum contact. On laminin or fibronectin and in the presence of growth factors, peripheral cells inside the halo of NBT-II aggregates did not exhibit desmosome linkages. These observations suggest that scatter effects per se are dependent on the composition of the extracellular matrix. In particular, on a substratum nonpermissive for direct cell translocation, individual cell dispersion can be replaced by en bloc patterns of migration following substratum conditioning by the cells.

The study of thin sections and replicas obtained after freeze-fracture showed that the intercellular junctions of the young secretory odontoblasts of the rat incisor were of two types. Zonula adherens (desmosomes) united the cellular bodies at the predentinal border. Ahead of these structures gap junctions were observed. An injection of vinblastine sulfate induced 4h, a) an enlargement of intermembranous spaces between the gap junctions, b) a reduction of the contact surfaces in the desmosomes and c) a thickening of the intracytoplasmic dense material made of microfilaments, associated with the junctional complex. These modifications favoured the observation of these structures after freeze-fracture. The desmosome junctions could play a mechanical function (partitioning of the predentinal compartment) whereas the gap junction allowed the electrotonic coupling of the odontoblasts. They allowed however an intercellular diffusion route for ionic components (calcium) and matrix components of serum origin.

In an attempt to induce acantholysis in cultivated normal human epidermis, porcine pancreatic elastase was used. After 30 minutes, the intercellular contact layers of desmosomes appeared discontinuous. Following the disappearance of the intercellular filamentous structures, desmosomes ruptured into two, and in 3 hours, typical acantholysis was observed. After 15 minutes, prior to the desmosomal changes, dermo-epidermal separation started at the subepidermal space. After 2 hours of elastase influence, anchoring filaments and the basal lamina were degraded. Five hours from the start of culture, the anchoring fibrils remained undigested by elastase. The mechanism of elastase influence on epidermis is assumed to depend rather on its proteolytic activity than on its elastolytic activity.

To analyse the effect of ethylenediamine tetraacetate (EDTA) on tumour cell adhesiveness, fine structure of intercellular junctions of rat ascites hepatoma cells AH136B and AH7974 (both forming cell islands in vivo) was first compared. The close contact of the apical portion of both cell islands was composed of tight junctions with a narrow gap. The close contact of the inner portion of AH136B cell islands was largely by simple apposition, while that of AH7974 cell islands had many intermediate junctions and desmosomes. Treatment with EDTA (2 mM) induced morphological alteration of simple apposition, intermediate junctions and desmosomes, but tight junctions remained intact. The effect of EDTA on such junctional complexes seemed to be partially reversible on readministration of Ca ions. Changes in desmosomes, as confirmed on AH7974 cells, were initiated by disappearance of the central disc of electron-dense materials, followed by marked opening of intercellular space and disappearance of endoplasmic laminar plaque. These results suggest that Ca ions may be concerned with maintaining the integrity of junctional complexes other than tight junctions.

During the postnatal period, cyclophosphamide produces in the retina of rats and mice rosettes containing malformed photoreceptor cells arranged in half-circle formations. The rosettes form only after injections given during the first 4 days post partum and only in the receptor layer which contains desmosomes. After this period, the retina becomes resistant to rosette formation. Cytological changes occur in the form of intense nuclear invaginations and membrane whorls, which may be regarded as compensatory processes.

The ultrastructural evaluation of the early alterations adriamycin-induced on cultured mice heart cells is reported. The major effects are hypertrophy of the sarcoplasmic reticulum and a market increase of the number and total extension of the gap junctions. These findings are discussed in the light of the information available in the literature.

Electron microscopy of the liver of methotrexate-treated psoriatic patients revealed junctional abnormalities consisting of detachment of desmosomal plaques between hepatocytes. Mitochondria anchoring desmosomal microfilaments were frequently noted.

Abnormal desmosomes were found in the endometrial epithelial cells of rats treated orally with gossypol acetic acid at a dose of 60 mg/kg/day for 30 days. Desmosomes in the endometrium of rats treated at 40 mg/kg/day were similar to those of controls. Abnormalities included an increase in desmosome number with prominent filamentous interconnections, asymmetry, disorganization of tonofilaments, missing tonofilaments, missing desmosomal plaques that did not appear to line up with those of the adjacent cell. It is thought that the antifertility actions of gossypol may be due to a disturbance of desmosome formation. This would cause a disruption of endometrial cell adhesion as well as an alteration in the microenvironment within cytoplasmic domains. These changes could lead to an endometrial environment unfavorable to fetal development.

Long-term repeated administration of isoproterenol (IPR) 2 mg/100 g bw, once daily for ten days, resulted in morphological changes in the intercellular junctions of rat submandibular glands, which were investigated by means of the freeze-fracture technique. A significantly increased number of tight-junctional strands was present. These junctional strands extended much deeper toward the basal membrane than those in normal acinar cells. The basal frontier strands that branched from the networks of tight junctions were elongated and had either free-endings or terminal loops, which were more frequently observed in the IPR-treated acinar cells than in untreated acinar cells. Some of the strands of tight junctions were connected to small gap junctions. The diameters of gap junctions were not significantly different from those of control acinar cells. However, smooth areas devoid of particles were found intermingling with the usual packed particles in irregularly shaped small gap junctions. There was no significant difference between the desmosomes of IPR-treated and untreated acinar cells, in terms of either morphology or distribution. These changes in junctional morphology in the IPR-treated acinar cells resemble those seen in salivary glands during development, and in some experimental conditions including tumorous changes.

Immature starfish oocytes are surrounded by envelopes consisting of follicular cells. These cells adhere to each other and to the oocyte, immobilizing the latter within the ovary. When isolated oocytes in their follicles are treated with 1-methyladenine (1-MeAde), germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) occur simultaneously. The 1-MeAde acts on the oocyte surface to produce a maturation-promoting factor (MPF) in the cytoplasm, which brings about GVBD. In the present study, MPF was found to induce FEBD as well as GVBD when injected into immature oocytes with their follicles in Asterina pectinifera. Although GVBD was induced by MPF in the presence of cytochalasin D, this drug prevented MPF-induced FEBD, and each follicular cell remained in situ on the surface of the oocyte. However, desmosomes connecting the processes of the follicle cell with the oocyte surface were disrupted following MPF injection even in the presence of cytochalasin D, and the processes became detached from the oocyte. FEBD occurred in these oocytes when cytochalasin D was removed, resulting in the formation of a small follicular clump by microfilament-mediated contraction of the follicle cells. These results show that FEBD is not brought about by the direct action of 1-MeAde but by the action of MPF. Therefore, in starfish, spawning as well as oocyte maturation is directly triggered by MPF produced under the influence of 1-MeAde.

To examine early intercellular junction changes caused by treatment with 9,10-dimethyl-1,2-benzanthracene (DMBA), rat lingual epithelium was cultivated in isolation and observed by electrophysiological, freeze-fracture and whole-mount electron microscopy. Electrophysiological measurements showed a transient decrease in membrane potential of -10.2 mV 6 h after the treatment. It returned to almost the same level as that of the control group 1 day later. Six hours after treatment, input resistance decreased rapidly to 5.3 M omega but increased to 18.0 M omega 12 h after treatment. Transient reduction of input resistance and membrane potential occurred prior to the decrease in the coupling ratio 6 h after treatment with DMBA. In freeze-fracture replicas, the number of gap junctions decreased by approximately 45% of the control value 6 h after treatment with DMBA. At 12 h and thereafter, the number and area of gap junctions subsequently decreased by 60-80% of the control value. Alterations in the number and area of desmosomes were similar to those of the gap junctions. The formation of epithelial cytoskeletons, partially devoid of the 2-4 and 5-8 nm filaments was also observed. A decrease in the density of filament networks beneath the plasma membranes was especially apparent. Treatment with a carcinogen brought about morphological cellular changes as early as 6 h after treatment, and such early changes might trigger metabolic cellular abnormalities. Affected cells appear to move away from normal cells in a process of repeated destruction and revision of intercellular junctions, and cytoskeletons.

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.

Beta-Aminopropionitrile (BAPN) retarded or suppressed epithelial changes in the medial edge of the palatal process in later stages of gestation in rats. Programmed cell death did not follow the usual pattern, and only a few lysosomes were observed on day 18 of gestation. The sensitivity of the medial epithelium to BAPN appeared to be different in various areas of the palatal epithelium; the epithelium on the anterior region of the palatal process was hypertrophied and keratinized, while posteriorly the medial or neighboring epithelium was very thin and, in neonatal rats, the covering was absent. A basal lamina was distinct in the anterior region and indistinct or fragmented posteriorly. Collagen fibers did not develop adjacent to the basal lamina, and an amorphous material was scattered throughout the mesenchymal tissue. These findings suggest that BAPN decreases the "connecting capacity" between mesenchyme and epithelium, and results in a modification of epithelial changes.

Recently, we have shown that transformation of human keratinocytes by SV40 virus induces the re-expression of characters found in fetal epidermis and monostratified epithelia. In the present study, TPA was found to alter some features of the human keratinocyte phenotype in a similar manner to SV40 transformation. Indeed, 12-O-tetradecanoyl-13-phorbol acetate (TPA) treatment induced the expression of cytokeratin no. 8, recognized by the monoclonal antibody TROMA-1, which is present in fetal epidermis and/or monostratified epithelia only, and fibronectin expression. However, TPA and SV40 transformation had specific non-overlapping effects. For example, TPA did not prevent terminal differentiation and stratification, as SV40-transformation did, but stimulated these processes. Moreover, TPA was found to induce additional changes in SV40-transformed keratinocytes. In particular it provoked the individualization of cells within the colonies. This effect was seen as little as 1 h after treatment and was reversible. This cellular alteration was accompanied by the reorganization of actin and by a decrease in the number of desmosomes; these changes were not observed after treatment of normal keratinocytes with TPA. These observations lead to the conclusion that TPA is able to trigger cellular responses which cannot be induced by SV40 products alone, but that TPA and some SV40 products can cooperate to elicit new responses, which could reflect a higher state of malignancy.

A continuous line derived from a human skin squamous cell carcinoma has been grown in media of high, normal and low Ca2+ concentrations. The growth rate was unaffected by the Ca2+ levels even though morphological changes were observed. Desmosomes were absent at low Ca2+ and areas of cell piling were observed at high Ca2+. Cell protein staining patterns on polyacrylamide gels were identical for cells grown at the three Ca2+ levels. The variations were minor for the glycoproteins reacted with 125I-conA. Lactoperoxidase iodination revealed changes in cell surface proteins, most markedly in the emergence of new proteins at high Ca2+.

Intact epithelial sheets were removed from rabbit corneas using Dispase II, a bacterial neutral protease. The freed sheets were placed on denuded corneal basal laminae and incubated at 35 degrees C for 3, 6, 18, or 24 h. Epithelial-basal lamina preparations were incubated in culture medium that either contained (a) varying concentrations of Ca2+ ions, (b) calmodulin antagonists, (c) exogenous calmodulin following an initial 6-h incubation in the presence of antagonists, or that lacked (d) Mg2+ ions. Tissues were processed for electron microscopy, and micrographs were taken of basal cell membranes. At least four experiments were conducted for each treatment, and for each experiment the total number of hemidesmosomes were counted along the basal membrane-basal lamina surface of eight cells. The number of hemidesmosomes formed was directly proportional to the increasing concentration of Ca2+. The presence of absence of Mg2+ ions did not change the numbers of hemidesmosomes formed. Calmodulin antagonists inhibited hemidesmosome formation, and this inhibition was reversed by the addition of calmodulin. Thus, hemidesmosome formation is Ca2+ dependent and appears to be mediated by a calmodulin-regulated mechanism.

The primary change found in cellular material expressed from open comedones of patients who had been treated with isotretinoin was disintegration of desmosomes. Consequently, there was lack of cohesion between cornified cells. A marked decrease in the quantity of sebaceous material and bacteria was also evident within the comedones.

We used antibodies raised against individual desmosomal components to study calcium-induced desmosome formation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the level of calcium ions in the culture medium, there is little contact between adjacent cells. Raising the level of calcium ions rapidly induces desmosome formation, and stratification occurs within 24 h. We found that before addition of calcium the 115,000- and 100,000-mol-wt core glycoproteins were distributed over the entire cell surface, whereas the plaque proteins (205,000 and 230,000 mol wt), the 82,000- and 86,000-mol-wt proteins, and the 150,000-mol-wt glycoprotein were located throughout the cytoplasm. 15 min after increasing the calcium ion concentration, all of these molecules appeared at the cell margins. The intensity of peripheral staining increased over the next 2 h and during this time the distribution of keratin filaments changed from predominantly perinuclear to extend throughout the cytoplasm. Keratinocytes could be dissociated with EDTA for up to 2 h after exposure to calcium. After 3 h of exposure to calcium the cells were no longer susceptible to EDTA dissociation and staining for desmosomal plaque antigens persisted in regions of intercellular contact. Desmosomal staining in stratified cultures became greatly reduced within 24 h of lowering the calcium ion concentration again. We have preliminary evidence that stratification occurs by breakdown of desmosomes at lateral surfaces and reformation at surfaces of contact between basal and suprabasal cells, rather than by rearrangement of existing desmosomes. Involucrin-positive cells in the monolayer appeared to contain more 205,000- and 230,000-mol-wt proteins free in the cytoplasm than involucrin-negative cells.

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.

Pieces of plantar stratum corneum were incubated with trypsin. This resulted in cell dissociation. The only observable ultrastructural change caused by trypsin was a degradation of desmosomal plates between dissociating cells. This suggests that desmosomes are of primary importance in plantar stratum corneum cell adhesion.

Neither stratifying (primary keratinocytes) nor simple (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney [MDBK]) epithelial cell types from desmosomes in low calcium medium (LCM; less than 0.1 mM), but they can be induced to do so by raising the calcium level to physiological concentrations (standard calcium medium [SCM], 2 mM). We have used polyclonal antisera to the major bovine epidermal desmosome components (greater than 100 kD) in a sensitive assay involving immunoprecipitation of the components from metabolically labeled MDCK cell monolayers to investigate the mechanism of calcium-induced desmosome formation. MDCK cells, whether cultured in LCM or SCM, were found to synthesize the desmosome protein, DPI and desmosome glycoproteins DGI and DGII/III with identical electrophoretic mobility, and also, where relevant, with similar carbohydrate addition/processing and proteolytic processing. The timings of these events and of transport of DGI to the cell surface were similar in low and high calcium. Although the rates of synthesis of the various desmosome components were also similar under both conditions, the glycoprotein turnover rates increased dramatically in cells cultured in LCM. The half-lives decreased by a factor of about 7 for DGI and 12 for DGII/III and, consistent with this, MDCK cells labeled for 48 h in SCM had three and six times the amount of DGI and DGII/III, respectively, as cells labeled for 48 h in LCM. The rate of turnover and the levels of DPI were changed in the same direction, but to much lesser extents. Possible mechanisms for the Ca2+-dependent control of desmosome formation are discussed in the light of this new evidence.

Retinoids, synthetic vitamin A analogs, stimulate mucous metaplasia and gap-junction proliferation in embryonic and neoplastic epidermis. Such effects demonstrate that vitamin A has potent effects on epidermal differentiation. Oddly, however, retinoid action in normal postnatal tissues, where these drugs are used clinically, appears to be quite different. In animals and humans, both topical and systemic retinoids produce acanthosis, hypergranulosis, and a relative (but not absolute) decrease in the thickness of the stratum corneum. These changes reflect the distinct boost in cell turnover that results from retinoid treatment. On the ultrastructural level, desmosomes are actively shed by cells of the spinous layer, resulting in many fewer attachment points along the cell membranes of the outer epidermis. Loss of desmosomes, coupled with decreased tonofilaments, enhanced keratinocyte autolysis, and intercellular deposition of glycoconjugates (not mucin), cause loosening and fragility of the stratum corneum (so-called anti-keratinizing effects). The biochemical basis of retinoid activity, in addition to stimulating increased cell turnover, appears to be a global enhancement of glycoconjugate synthesis and the generation of less mature keratins. The epidermal effects of retinoids can be exploited therapeutically: to cause loosening of thickened stratum corneum, for example in psoriasis or ichthyosis; to enhance penetration of pharmacologic agents such as 5-fluorouracil across hypertrophic actinic keratoses; and to normalize differentiation in neoplastic epidermis involving mucous metaplasia and gap-junction proliferation.

The processes of desmosome formation and keratinization were studied in isolated frog skins cultured in a two-compartment (mucosal and serosal) chamber. Before culture, the skin fragments were trypsinized (stratum corneum together with some parts of stratum granulosum and spinosum were scraped off with forceps) allowing the stratum germinativum to remain on the dermis. When both the mucosal and the serosal culture media contained 1.5 mM calcium and 86 mM sodium concentrations, fully developed desmosomes were differentiated and no keratinization occurred. When the mucosal medium was lowered in two steps to a final calcium concentration of 0.5 mM by dilution with tridistilled sterile water, poorly developed desmosomes were formed, keratinocytes interdigitated and the keratinization was strongly enhanced. The calcium-dependent desmosome formation was affected by the salt gradient established across the skin. These two effects, modulated desmosome formation (calcium) and increased keratinization (sodium), were concomitant with but did not complement one another.

The filamentous cytoskeletons of epidermal cells of the bullfrog (Rana catesbeiana) were investigated by electron microscopy. Following treatment with trypsin, sheets of epithelium were removed from swatches of abdominal skin. Trypsinization produces differential effects on the ultrastructure of the various cell layers. The desmosomes of all layers, except those of the stratum corneum, are split by trypsinization and the resulting desmosomal plaques fastened to tonofilaments are retracted into cells to form deep "inpouchings" of the plasma membranes, while tonofilament bundles become diffuse. Epidermal sheets were gently homogenized to form a suspension of cell remnants with damaged plasma membranes as indicated by vital dye exclusion tests and electron microscopy. Cytoskeletons retain their shapes, yet the lateral distances between individual tonofilaments within bundles appear to increase, thus forming diffuse lacelike structures. These observations support the suggestion that tonofilament bundles, when fastened to desmosomes, have elastic properties. The possible role of the cytoskeletons in the maintenance of cell size and shape in an ion-transporting epithelium is discussed.

Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin-containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes.

Although much recent work suggests that retinoids can prevent the development of epithelial cancers, their mechanism of action remains unknown. Since malignancy has been associated with alterations in gap junctions, desmosomes, microfilaments, and hemidesmosomes, the authors examined freeze-fracture replicas and thin sections of cell membranes of: (1) 11 basal cell cancers (BCC) treated twice daily for two weeks with topical 1.0% retinoid acid (RA); (2) 21 BCC treated for 2 to 17 weeks with oral 13-cis retinoic acid (CRA) (1.0-8.0 mg/kg/day); and (3) 17 BCC prior to retinoid treatment and/or after applications of vehicle alone. Both thin sections and replicas were examined and photographed in a single-blind fashion, and the density and size distribution of gap junctions and desmosomes were computed planimetrically. Topical RA treatment induced a two-fold increase in gap junction density (P less than 0.025) over controls. In contrast, RA produced a concurrent = 35% decrease in desmosome density. Systemic CRA did not significantly alter either gap junction or desmosome density or size. Finally, neither RA nor CRA treatment appeared to influence hemidesmosome or microfilament populations. Structural changes in both treatment groups did not correlate with either tumor regression or inflammation. Topical and systemic retinoids may exert their antineoplastic activity by different cellular mechanisms.

Changes in desmosomes were noted in nonacantholytic regions of skin explants cultured in the presence of intercellular antibodies. These changes included dissociation of desmosomes, as well as a "selective loss" of desmosomal components in one or both of neighboring cells.

The tracheal epithelium of a variety of laboratory species is widely used as a model system in studies of epithelial biology and respiratory carcinogenesis. The purpose of this study was to evaluate the response of the tracheal epithelium to cytotoxic injury in a primate species that may have an epithelium more representative of that in man than smaller laboratory species. This study evaluated changes in the light-microscopic, surface, and ultrastructural appearance of the tracheobronchial epithelium of bonnet monkeys exposed for 3 or 7 days to 0.64 ppm ozone. Population densities, epithelial volumetric densities, and thymidine labeling indexes were determined for cells from posterior membranous and anterior cartilaginous trachea and mainstem bronchus. Ozone-induced epithelial changes were characterized by decreased numbers of ciliated cells, loss of cilia, and necrosis of ciliated cells. There were alterations in mucous (goblet) cell granules. There was an increase in extracellular space and focal epithelial stratification that was associated with increased numbers of small mucous granule cells and the presence of an epithelial cell type not seen in control animals (intermediate cells). There was an increase in cytoplasmic filaments and desmosomal attachments in basal cells, small mucous granule cells, and intermediate cells. Regional differences in lesion distribution were demonstrated by scanning electron microscopy. Longitudinal streaks of ciliary loss were evident in posterior membranous trachea, but ciliary loss in the ventral trachea was most prominent over the posterior border of the cartilaginous rings. The thymidine labeling index and numbers of necrotic ciliated cells were greater after 3 days than after 7 days of continuous exposure. Foci of stratification were often associated with increased numbers of labeled nuclei in the suprabasal region of the epithelium. The results of this study suggest that small mucous granule cells and intermediate cells are important participants in the repair of chemically injured airway epithelium; stratification and increased amounts of cytoplasmic filament bundles and desmosomal attachments, rather than being evidence of squamous metaplasia or dysplastic change, might be stereotypic responses of airway epithelium to injury; and the ciliated cell population becomes less susceptible to ozone-induced necrosis with continuing exposure.