PCC4azal teratocarcinoma stem cell differentiation in culture: III. Cell-to-cell communication properties

The cell-to-cell communication properties of the PCC4azal embryonal carcinoma cells and their differentiated endoderm-like cells and giant cells were characterized by ionic coupling, metabolic coupling, and transfer of an injected fluorescein dye. All PCC4azal cell types communicate well with one another: stem cells with stem cells, endoderm-like cells with endoderm-like cells, and giant cells with giant cells. In addition, the stem cells communicate well with giant cells as assayed by metabolic coupling. The ability of the undifferentiated teratocarcinoma cells to metabolically couple with a variety of heterologous cell types was examined. The stem cells were always efficient donors but very poor recipients in almost all combinations with cells of fibroblast or epithelial morphology that were derived from several different species. In contrast, these same heterologous cell types were both efficient donors and efficient recipients with each other.     

PCC4azal teratocarcinoma stem cell differentiation in culture: I. Biochemical studies

PCC4azal embryonal carcinoma cells were observed to spontaneously differentiate under defined culture conditions to endoderm-like cells and subsequently to giant cells. This differentiation was examined by determining the specific activities of several enzymes in the stem and endoderm-like cell populations. With differentiation, the level of alkaline and acid phosphatase activities remained unchanged, plasminogen activator specific activity increased fivefold, and lactate dehydrogenase (LDH) specific activity decreased to 40% of its original level. Isozyme analysis revealed a shift of the LDH isozymes toward LDH₁ with the appearance of LDH₂ for the first time in the endoderm-like cells. The surface antigen SSEA-1 was detected by indirect immunofluorescence on virtually all of the stem cells. However, the SSEA-1 antigen was not present on many of the endoderm-like cells, and it was completely undetectable on giant cells as assayed by immunofluorescence. The expression of H-2 antigen was examined in a similar manner using anti-H-2^b antiserum; this antigen was not detected on the stem, endoderm-like, or giant cells. Thus, there are defined biochemical changes that accompany the differentiation of PCC4azal stem cells in culture.     

Teratocarcinoma X friend erythroleukemia cell hybrids resemble their pluripotent embryonal carcinoma parent.

Five independent clones of somatic cell hybrids have been produced by fusing FBU Friend erythroblastic leukemia cells with cells of the pluripotent teratocarcinoma-derived embryonal carcinoma line PCC4azal. All five lines closely resemble their PCC4azal parent. They look like embryonal carcinoma cells by phase contrast and electron microscopy, have high levels of alkaline phosphatase but low levels of acetylcholinesterase, and, like PCC4azal, express both LDH-A and LDH-B. Tumors produced from hybrid lines often contain large amounts of differentiated tissue, including representatives of all three of the classical germ layers. These results suggest that the genome of a pluripotent mammalian cell, far from being unconditionally susceptible to whatever signals differentiated cells employ to maintain their stable phenotype, may itself be able to “reset” the genome of the differentiated cell.     

Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes

Junctional complexes such as tight junctions, adherens junctions, and desmosomes play crucial roles in the structure and function of epithelial cells. These junctions are involved in increasing cell-cell contact and as well serve as signaling centers regulating multiple functions in epithelial cells. Carcinoma cell lines cultured in the laboratory generally lack junctional complexes. However, studies directed towards understanding the distribution of junctional complexes in human cancer tissues are lacking. In this study, we analyzed by electron microscopy the distribution of junctional complexes in patients diagnosed with renal clear-cell carcinoma. We found that both tight junctions and adherens junctions were drastically reduced in patients with cancer compared to normal tissues. Desmosomes were not detected in normal proximal tubules while distinctly present in cancer tissues. These results suggest that analysis of junctional complexes in human tumors should provide valuable information that might have prognostic and diagnostic value.     

Contacts between pigmented retina epithelial cells in culture.

The behaviour of primary cultures of dissociated embryonic chick pigmented retina epithelial (PRE) cells has been investigated. Isolated PRE cells have a mean speed of locomotion of 7-16 mum/h. Collisions between the cells normally result in the development of stable contacts between the cells involved. This leads to a gradual reduction in the number of isolated cells and an increase in the number of cells incorporated into islands. Ultrastructural observations of islands of cells after 24 h in culture show that junctional complexes are present between the cells. These complexes consist of 2 components: (a) an apically situated region of focal tight junctions and/or gap junctions, and (b) a more ventrally located zonula adhaerens with associated cytoplasmic filaments forming a band running completely around the periphery of each cell. The intermembrane gap in the region of the zonula is 6-0-12-0 nm. The junctional complexes become more differentiated with time and after 48 h in culture consist of an extensive region of tight junctions and/or gap junctions and a more specialized zonula adhaerens. It is suggested that the development of junctional complexes may be responsible for the stable contacts that the cells display in culture.     

Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis

Endothelial cells lining the vessel wall are connected by adherens, tight and gap junctions. These junctional complexes are related to those found at epithelial junctions but with notable changes in terms of specific molecules and organization. Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis. In this review, we will focus on specific mechanisms of endothelial tight and adherens junctions.     

Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis

Endothelial cells lining the vessel wall are connected by adherens, tight and gap junctions. These junctional complexes are related to those found at epithelial junctions but with notable changes in terms of specific molecules and organization. Endothelial junctional proteins play important roles in tissue integrity but also in vascular permeability, leukocyte extravasation and angiogenesis. In this review, we will focus on specific mechanisms of endothelial tight and adherens junctions.     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.     

Molecular anatomy of interendothelial junctions in human blood-brain barrier microvessels

Immunogold cytochemical procedure was used to study the localization at the ultrastructural level of interendothelial junction-associated protein molecules in the human brain blood microvessels, representing the anatomic site of the blood-brain barrier (BBB). Ultrathin sections of Lowicryl K4M-embedded biopsy specimens of human cerebral cortex obtained during surgical procedures were exposed to specific antibodies, followed by colloidal gold-labeled secondary antibodies. All tight junction-specific integral membrane (transmembrane) proteins--occludin, junctional adhesion molecule (JAM-1), and claudin-5--as well as peripheral zonula occludens protein (ZO-1) were highly expressed. Immunoreactivity of the adherens junction-specific transmembrane protein VE-cadherin was of almost similar intensity. Immunolabeling of the adherens junction-associated peripheral proteins--alpha-catenin, beta-catenin, and p120 catenin--although positive, was evidently less intense. The expression of gamma-catenin (plakoglobin) was considered questionable because solitary immunosignals (gold particles) appeared in only a few microvascular profiles. Double labeling of some sections made possible to observe strict colocalization of the junctional molecules, such as occludin and ZO-1 or JAM-1 and VE-cadherin, in the interendothelial junctions. We found that in human brain microvessels, the interendothelial junctional complexes contain molecular components specific for both tight and adherens junctions. It is assumed that the data obtained can help us find the immunodetectable junctional molecules that can serve as sensitive markers of normal or abnormal function of the BBB.     

Effects of anti-embryonal carcinoma serum on aggregation and metabolic cooperation between teratocarcinoma cells

Effects of rabbit anti-embryonal carcinoma IgG on embryonal carcinoma cells and their differentiated derivatives were studied at different levels of cell-cell interaction. Fab fragments of anti-EC IgG were found to inhibit aggregation of the majority of EC cell lines. Two, however, were insensitive. Anti-EC Fab fragments act also on the transfer of metabolites between EC cells: the rescue of HPRT^? EC cells by HPRT⁺ EC cells in selective medium is abolished. These findings are correlated with the disappearance of tight and gap junctions from the surface of EC cells (Dunia et al., 1979). The presence of the surface structure involved in the action of anti-EC Fab fragments was tested by absorption experiments followed by decompaction test on PCC4 Aza R1 cells. All EC cell lines and two embryonic cell lines—a trophectodermal and an endodermal line—were found to bear material absorbing the decompacting activity. The results are discussed in terms of state of differentiation of the cell lines and of complexity of aggregation of embyronic cells.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

The intercellular junctions of guinea-pig placental capillaries: a possible structural basis for endothelial solute permeability

The endothelial cell junction in guinea-pig placental capillaries consists of a continuous ribbon desmosome (zonula adherens) within which lies a particulate tight junction consisting of between one and five anastomosing strands. The intercellular space at these tight junctions is narrowed and is subdivided by junctional bars which are probably continuous with the intramembrane particle rows seen in freeze-fracture replicas of the junctions. Perfusion with lanthanum salts shows the gaps between the junctional bars to be lanthanum-filled and the entire junction to be lanthanum permeable. The estimated size of the spaces between the junctional bars is consistent with the junctional pore size indicated by previous ultrastructural tracer studies. The wider lateral intercellular space of the ribbon desmosome is spanned by more widely spaced "linkers" which may act as a coarser three-dimensional filter in series with size-limiting pores between the tight junctional bars.     

Localization of phosphatase activities on mouse teratocarcinoma embryoid bodies

Non-specific alkaline phosphatase and Mg²⁺-dependent adenosine triphosphatase activities were ultracytochemically investigated on embryoid bodies of murine teratocarcinomas, in order to find markers of endodermal cell differentiation of early embryonic cells. The former was localized mainly on the cell surface of inner embryonal carcinoma cells, as already shown by other workers, and weakly on the bound surface of outer endodermal cells of embryoid bodies. The latter, however, was found only on the outer free surface of endodermal cells and never on the surface of embryonal carcinoma cells. It suggests that Mg²⁺-dependent ATP activity might become the marker for early differentiation of embryonal carcinoma cells.     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Teratocarcinoma differentiation: plasminogen activator activity associated with embryoid body formation.

Changes in plasminogen activator activity have been examined as a clonal line of mouse embryonal carcinoma cells aggregate and differentiate to form cystic embryoid bodies in vitro. Within the first 10 days of study, the pluripotent embryonal carcinoma cells aggregate; a layer of endodermal cells appears on the outside of the aggregate forming an embryoid body; a basement membrane forms between the outer layer of endodermal cells and the internal cells; a cyst forms within the embryoid body; and the internal cells assume a columnar appearance along the inner portion of the basement membrane. After the formation of the endodermal layer, there is a rise in intracellular plasminogen activator activity. This rise continues for up to 25 days in culture, providing that the three-dimensional integrity of the embryoid bodies is maintained by culturing them on bacterial petri dishes. Selective removal of the outer endodermal layer of cells reduces the plasminogen activatory activity of the resulting embryoid body cores. Intracellular and secreted plasminogen activator activity of simple embryoid bodies composed of only two cell types can be increased by culturing the embryoid bodies in dbcAMP, theophylline, or cholera toxin. These results suggest that the embryoid body endodermal cells are the source of a cAMP-inducible plasminogen activator activity.     

Reciprocal influence of connexins and apical junction proteins on their expressions and functions

Membranes of adjacent cells form intercellular junctional complexes to mechanically anchor neighbour cells (anchoring junctions), to seal the paracellular space and to prevent diffusion of integral proteins within the plasma membrane (tight junctions) and to allow cell-to-cell diffusion of small ions and molecules (gap junctions). These different types of specialised plasma membrane microdomains, sharing common adaptor molecules, particularly zonula occludens proteins, frequently present intermingled relationships where the different proteins co-assemble into macromolecular complexes and their expressions are co-ordinately regulated. Proteins forming gap junction channels (connexins, particularly) and proteins fulfilling cell attachment or forming tight junction strands mutually influence expression and functions of one another.     

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.     

Some observations on the intercalated disc of a rat cardiac muscle.

During the experimental investigations special attention was paid to the orthomorphology of the ultrastructure of cardiac muscle intercalated disc in a rat. The contact junction between the cellular membranes of adjacent cells and intercellular spaces are typical for the cardiac muscle intercalated disc. Attention was paid to the three zone system of the junctions, namely: 1. nexus (zonula occludens), 2. fascia adherens (zonula adherens) and 3. macula adherens (similar to the desmosome). Apart from this the cell membranes adjacent to the cells may form digital indentations. A single injection of adrenaline in a dose of 2.4 mg/kg causes sinuate widenings of the intercellular space, but only in the zone of the fascia adherens. The structure of the nexuses and maculae adherentes is unchanged during this time.