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STUDY QUESTION: DO maternal endometrial epithelial cell (EEC) differentiation and polarity impact the invasive capacity of extravillous trophoblast (EVT) cells during early human implantation? SUMMARY ANSWER: In a three dimensional (3D) confrontation co-culture the invasiveness of the human trophoblast cell line AC-1 M88was inversely correlated with the degree of differentiation and polarization of human endometrial adenocarcinoma cell spheroids. WHAT IS KNOWN ALREADY: In a previous study desmosomal and adherens junction proteins were shown to spread from a subapically restricted lateral position to the entire lateral membrane in human glandular EECs during the implantation window of the menstrual cycle. Whether this change in EEC junction localization has an impact on the interaction of EVT cells with glandular EECs during early human implantation is not known. STUDY DESIGN, SIZE, DURATION: A new 3D cell culture system was developed in order to mimic early implantation events in humans. As a model for the invasion of endometrial glands by EVT cells, spheroids of three differently differentiated and polarized endometrial adenocarcinoma cell lines were confronted with an EVT cell line in co-culture experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three human adenocarcinoma EEC lines were chosen for this study because of their differences in differentiation and polarization: HEC-1 -A, which is well differentiated and highly polarized, Ishikawa, which is well differentiated and moderately polarized, and RL95-2, which is moderately differentiated and poorly polarized. When the cell lines were grown in reconstituted basement membrane, they formed gland-like, multicellular spheroids. The degree of polarization within the different EEC spheroids was assessed by 3D confocal immunofluorescence microscopy detecting the basal membrane protein integrin a6, the apical tight junction-associated protein ZO-1 and the desmosomal plaque protein desmoplakin 1 /2 (Dsp). Cells of the human EVT cell line AC-1 M88, which is a fusion cell line of primary EVT cells and choriocarcinoma-derivedJEG-3 cells, were added to the different EEC spheroids to examine their interaction. For the analyses of trophoblast-endometrial confrontation sites, HLA-G was used as a specific EVT cell marker. MAIN RESULTS AND THE ROLE OF CHANCE: The endometrial HEC-1 -A and Ishikawa cells formed gland-like structures in reconstituted basement membrane with apicobasal polarization towards their well-developed internal lumina, while most of the RL95-2 spheroids showed no lumen formation at all. The three EEC lines strongly differed in their apicobasal distribution pattern of Dsp. Ishikawa and HEC-1 -A spheroids showed a subapical concentration of Dsp. In contrast, an equal distribution of Dsp was discerned along the entire lateral membranes in RL95-2 spheroids. In 3D confrontation co-cultures the highest invasiveness of AC-1 M88 was observed in the poorly polarized RL95-2 spheroids. LIMITATIONS, REASONS FOR CAUTION: Human endometrial and trophoblast cell lines were used for this study because of ethical and legal restrictions for implantation studies with human blastocysts and because of limited access to primary human endometrial cells. WIDER IMPLICATIONS OF THE FINDINGS: The presented 3D cell culture system can be used to investigate the contribution of epithelial junctions to trophoblast-endometrial interactions. The identified impact of endometrial differentiation and polarity on the invasiveness of EVT cells improves our understanding of the relevance of endometrial receptivity for early implantation and may contribute to higher success rates in assisted reproductive technology. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grant 146/14, 'START-Program', Medical Faculty, RWTH Aachen University, to V.U.B., by Grant Lec_16_12, 'RWTH Lecturer Award', RWTH Aachen University to I.C.-L and by the German Research Council (Grant LE 566-20-1). The authors declare no conflict of interest. [ABSTRACT FROM AUTHOR]

Background: Epidermal differentiation is a multilevel process in which keratinocytes need to lose their organelles, including their mitochondria, by autophagy. Disturbed autophagy leads to thickening of the epidermis as seen in pachyonychia congenita (PC), a rare skin disease caused by mutations in keratins 6, 16 and 17., Objectives: To ask if mitophagy, the selective degradation of mitochondria by autophagy, is disturbed in PC and, if so, at which stage., Methods: Immortalized keratinocytes derived from patients with PC were used in fluorescence-based and biochemical assays to dissect the different steps of mitophagy., Results: PC keratinocytes accumulated old mitochondria and displayed disturbed clearance of mitochondria after mitochondrial uncoupling. However, early mitophagy steps and autophagosome formation were not affected. We observed that autolysosomes accumulate in PC and are not sufficiently recycled., Conclusions: We propose an influence of keratins on autolysosomal degradation and recycling. What's already known about this topic? Terminal epidermal differentiation is a multistep process that includes the elimination of cellular components by autophagy. Autophagy-impaired keratinocytes have been shown to result in thickening of epidermal layers. Hyperkeratosis also occurs in pachyonychia congenita (PC), a rare skin disease caused by mutations in keratins 6, 16 and 17. What does this study add? Keratins contribute to mitochondrial quality control as well as maintenance of mitochondria-endoplasmic reticulum contact sites. Keratins influence autolysosomal maturation or reformation. What is the translational message? Overaged mitochondria and autolysosomes accumulate in PC. Mutations in keratin 6a lead to severely impaired mitophagy, which might contribute to PC pathogenesis., (© 2019 British Association of Dermatologists.)

Intermediate filaments are major phosphoproteins. The complex patterns of intermediate filament phosphorylation make up a poorly understood code reflecting cytoskeletal properties and cellular function through an intense crosstalk with multiple signaling pathways. This review focuses on the epithelial keratin intermediate filaments highlighting the tight-knit relationship of keratin phosphorylation and network organization during cell division and apoptosis, and the importance of keratin phosphorylation during epithelial stress responses. The occurrence of keratin phosphorylation in genetic skin diseases and acquired diseases of simple epithelial tissues in liver, pancreas, and colon will be discussed. Finally, we will review the role of keratin phosphorylation in cancer with an emphasis on migration., (© 2017 Elsevier Inc. All rights reserved.)

The tetraspan membrane proteins of the synaptogyrin and synaptophysin type are abundant and evolutionary conserved synaptic vesicle membrane proteins whose functions are poorly defined. Their depletion does not interfere with proper neuronal development and basic neuronal function. In the search for their function we use the genetic model organism Caenorhabditis elegans in which, in contrast to vertebrates, the synaptogyrin but not the synaptophysin orthologue is predominant in neurons. Employing fluorescent reporter constructs we find that synaptogyrin is expressed in all GABAergic neurons and most, though not all other neurons. Subjecting animals either lacking or overexpressing synaptogyrin to the epileptogenic GABA antagonist pentylenetetrazole reveals increased sensitivity in comparison to the wild-type. Detailed analyses further uncover mildly altered motility, slightly reduced sensitivity to the acetylcholine esterase inhibitor aldicarb and decreased recruitment of synaptobrevin but not of RAB-3 to synapses. Furthermore, synthetic phenotypes are observed with mutants of the synaptic vesicle recycling machinery, notably with synaptotagmin, synaptojanin and endophilin rather than with mutants involved in clathrin-dependent endocytosis. Taken together, these observations assign a distinct modulatory and redundant neuronal function to synaptogyrin., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

The integral membrane protein synaptophysin is one of the most abundant polypeptide components of synaptic vesicles. It is not essential for neurotransmission despite its abundance but is believed to modulate the efficiency of the synaptic vesicle cycle. Detailed behavioral analyses were therefore performed on synaptophysin knockout mice to test whether synaptophysin affects higher brain functions. We find that these animals are more exploratory than their wild type counterparts examining novel objects more closely and intensely in an enriched open field arena. We also detect impairments in learning and memory, most notably reduced object novelty recognition and reduced spatial learning. These deficits are unlikely caused by impaired vision, since all electroretinographic parameters measured were indistinguishable from those in wild type controls although an inverse optomotor reaction was observed. Taken together, our observations demonstrate functional consequences of synaptophysin depletion in a living organism.

Synaptophysin (SYP) is a major protein of neurotransmitter-containing vesicles spanning the membrane four times and contributing to various aspects of the synaptic vesicle cycle. The split-ubiquitin yeast two-hybrid system was used to characterize molecular interactions of membrane-bound, full-length murine SYP. In this way, the known homophilic SYP-SYP association could be confirmed and heterophilic binding of SYP to other tetraspan vesicle membrane proteins of the secretory carrier-associated membrane- and synaptogyrin-type could be detected for the first time. SYP-binding was also observed for the vSNARE synaptobrevin2 and various membrane and membrane-associated proteins. Double labeling immunofluorescence microscopy of murine retina, co-immunoprecipitation experiments and fluorescence energy resonance transfer (FRET) analyses between fluorescent protein-tagged polypeptides were carried out to validate and further characterize the association of SYP with the tetraspan vesicle membrane proteins secretory carrier-associated membrane protein 1 and synaptogyrin3, with synaptobrevin2, and the newly identified binding partners phospholipase D4, stathmin-like3, Rho family GTPase2 and ADP-ribosylation factor interacting protein2. It was observed that the carboxyterminus of SYP is dispensable for association with integral membrane proteins while it is needed for binding to membrane-associated polypeptides. The latter appears to be regulated by phosphorylation, since src homology 2-domains were shown to attach to the multiple carboxyterminal phosphotyrosine residues of SYP. In conclusion, the association of SYP with different tetraspan vesicle membrane proteins suggests shared functions and the multiple other interactions identify SYP as part of a membrane platform acting as a facilitator of various steps of the synaptic vesicle cycle.

We have previously described vulva carcinoma-derived A-431 subclone AK13-1, which stably expresses fluorescently labeled cytokeratin filaments (CKFs). Time-lapse fluorescence microscopy of these cells permits the continuous monitoring of the dynamics of the CKF cytoskeleton in vivo. To study mechanisms and principles of CKF disassembly as it occurs, e.g., during mitosis and liver disease, we have treated cells with the phosphatase inhibitor okadaic acid (OA), which induces complete CKF network breakdown within 3-5 h without significantly affecting the organization of the actin- and tubulin-based cytofilaments. In time-lapse movies, we find that the network breakdown starts at the cell periphery and proceeds toward the cell center, where residual filaments become compacted into a prominent perinuclear ring. The progressing disassembly is paralleled by an increase of diffuse fluorescence throughout the cytoplasm and the appearance of non-filamentous spheroidal aggregates. They are formed in the filament-free cell periphery from non-filamentous precursors and can sometimes be detected in the proximity of desmosomes. Other aggregates are either found in close apposition to CKFs or are generated directly from the compacted perinuclear material. Primary granules later fuse, thereby producing structures of considerable size. We show that CKF network breakdown and granule formation rely on metabolic energy and that the continued presence of OA is needed for its completion. We conclude that phosphorylation/dephosphorylation is an important mechanism regulating CKF network dynamics in vivo with far-reaching implications for the understanding of epithelial plasticity and pathology.

Of the three major cytoskeletal filament systems, the intermediate filaments are the least understood. Since they differ fundamentally from the actin- and microtubule-based networks by their lack of polarity, it has remained a mystery how and where these principally endless filaments are formed. Using a recently established epithelial cell system in which fluorescently labeled intermediate filaments of the cytokeratin type can be monitored in living cells, we address these issues. By multidimensional time-lapse fluorescence microscopy, we examine de novo intermediate filament network formation from non-filamentous material at the end of mitosis and show that it mirrors disassembly. It is demonstrated that filament formation is initiated from the cell cortex without focal preference after cytokinesis. Furthermore, it is shown that this process is dependent on energy, on the integrity of the actin filament network and the microtubule system, and that it can be inhibited by the tyrosine phosphatase inhibitor pervanadate. Based on these observations, a two-step working model is proposed involving (1) interactions within the planar cortical layer acting as an organizing center forming a two-dimensional network and (2) subsequent radial dynamics facilitating the formation of a mature three-dimensional network., (Copyright 2001 Wiley-Liss, Inc.)

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.

The N-glycosylated integral membrane protein synaptophysin is one of the major polypeptide components of small presynaptic transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells of mammals. Functional properties, including a possible participation in channel formation, have been investigated by integration of purified synaptophysin into planar lipid bilayers. To overcome some of the inherent limitations of such an in vitro approach we have overexpressed the rat synaptophysin cDNA in nonneuronal, non-neuroendocrine insect cells with the help of recombinant baculovirus. The complete polypeptide was produced in infected ovarian Sf9 cells at levels exceeding those observed in rat brain. The partially N-glycosylated molecules could be extracted from membranes with non-ionic detergents, most effectively with n-octyl-beta-D-glucopyranoside, and could be enriched on chromatofocusing columns. By immunoelectron microscopy synaptophysin was shown to be integrated in the correct orientation into the endoplasmic reticulum, various pleomorphic vesicles and the plasma membrane. Using cell fractionation, including density gradient centrifugation and immunoisolation, we characterized distinct synaptophysin-rich vesicles. These vesicles may help to understand molecular principles of vesicle biogenesis in general and the function of synaptophysin in particular.

Synaptophysin is one of the major integral membrane proteins of the small (30-50nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. The behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.

The acid cytokeratin K17 is inducible by interferon-gamma (IFN-gamma), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN-gamma in epithelial cells. The 5'-flanking region of the K17 gene (positions -1762 to -13), cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN-gamma but not IFN-alpha in transient transfection assays. Sequence analysis revealed three putative gamma-interferon activation sites (GAS). Band-shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions -1528 to -1515) was found to bind GAF/STAT91 and to compete with tryptophanyl-tRNA synthetase (IFP53/WRS)-GAS for binding to GAF; in contrast, GAS1 (positions -183 to -171) and GAS2 (positions -290 to -277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5'-flanking region (positions -1762 to -111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GAS1 element rendered the promoter uninducible by IFN-gamma. Surprisingly, transfection of a CAT-reporter gene construct harbouring a promoter segment (positions -111 to +13) devoid of the GAS elements revealed enhanced CAT-gene expression upon IFN-gamma treatment. The interaction of GAS1 with the interferon-responsive promoter region in the physiological context remains to be clarified.

We have previously described the three loci CK-CA, CK-CB and CK-CC in the human genome that contain clustered type-I cytokeratin genes and reported the complete nucleic acid sequences of the functional cytokeratin 17 gene located in CK-CA and two closely related pseudogenes present in CK-CB and CK-CC [Troyanovsky, S.M., Leube, R.E. & Franke, W.W. (1992) Eur. J. Cell Biol. 59, 127-137]. By nucleic acid sequence analysis, we now show that extensive similarities between the functional gene and the pseudogenes exist in the 5'-upstream region. However, despite the high degree of nucleic acid identity (94%), only the 5'-upstream region of the functional gene was able to induce significant transcriptional activity in transfected cells of epithelial origin. Using chimeric upstream regions consisting of different fragments from the pseudogene and the functional gene, we made the surprising observation that cis elements in the proximal 5'-upstream region of the pseudogene promoter can cooperate with distal enhancer elements of the functional gene to induce strong chloramphenicol-O-acetyltransferase activity in transfected HeLa cells. A major site in the proximal upstream region was identified by deoxyribonuclease protection experiments to be necessary for this cooperative effect. The structure and properties of this element were further analysed by transfection of different chloramphenicol-O-acetyltransferase gene constructs, and by nucleic acid sequence comparison to corresponding regions of the related cytokeratins 14 and 16. It is concluded that the upstream regions identified in this study contribute to the strong expression of the human cytokeratin 17 gene in a coordinated fashion.

The integral membrane protein synaptophysin is one of the major polypeptide components of the small, electron-translucent, transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine (NE) cells. In an attempt to identify synaptophysin-related molecules, such as synaptoporin, it was noticed in polymerase chain reaction (PCR) experiments that products having the expected size could be amplified not only from neuronal and NE cells, but also from non-NE cells. Northern blot hybridization analyses demonstrated that certain non-NE cells express low amounts of synaptophysin mRNA although the encoded polypeptide could not be detected. These observations, however, did not explain the consistent amplification of cDNA fragments regardless of cell type. PCR products were therefore cloned and a novel type of cDNA was identified in rat and human. The partial human cDNA was completed by isolation of phage cDNA clones constructed from a human keratinocyte cell line (HaCaT) and by PCR. When used in hybridization experiments with genomic DNA, this clone recognized a single gene. The 2106 bp cDNA contains an open reading frame coding for a polypeptide of calculated molecular weight 28,565 and having an isoelectric point of 8.45. This polypeptide is very similar to synaptophysin in the four transmembrane domains and the connecting loop regions but lacks the characteristic cytoplasmic tail. Extensive PCR analyses and Northern blot hybridization experiments demonstrated that the synaptophysin-related gene is ubiquitously expressed in vitro and in vivo. To stress the ubiquity of expression in contrast to the restricted distribution of synaptophysin and synaptoporin, I propose to refer to the encoded polypeptide as pantophysin.

To examine the potential of cytoplasmic portions ("tails") of desmosomal cadherins for assembly of desmosome plaque structures and anchorage of intermediate filaments (IFs), we transfected cultured human A-431 carcinoma cells, abundant in desmosomes and cytokeratin IFs, with constructs encoding chimeric proteins in which the transmembranous region of connexin 32 had been fused with tails of desmocollin (Dsc) or desmoglein (Dsg). The results show that the tail of the long splice form a of Dsc, but not its shorter splice form b, contains sufficient information to recruit desmoplakin and plakoglobin to connexon membrane paracrystals (gap junctions) and to form a novel kind of plaque at which cytokeratin IFs attach. By contrast, chimeras containing a Dsg tail, which accumulated in the plasma membrane, showed a dominant-negative effect: they not only were unable to form gap junction structures and plaques but also led to the disappearance of all endogenous desmosomes and the detachment of IFs from the plasma membrane.

Expression of keratins 1, 6, 15, 16, and 20 was examined in normal cervical epithelia, squamous metaplasia, various grades of cervical intraepithelial neoplasia, and both squamous cell carcinomas and adenocarcinomas of the cervix with monospecific antibodies. Ectocervical epithelium contains all of these keratins except keratin 20. They show a heterogeneous distribution, with a basally restricted detection of keratin 15. Endocervical columnar cells were found to contain significant amounts of keratin 16, whereas the subcolumnar reserve cells expressed considerable amounts of keratin 15 and 16, and frequently keratin 6. These reserve cell keratins were also found in immature and mature squamous metaplastic epithelium. In the cervical intraepithelial neoplastic lesions they were generally found with increasing intensity as the severity of the lesion progressed. In the keratinizing variety of squamous cell carcinoma of the cervix, these three keratins seem to constitute an important part of the intermediate filament cytoskeleton, whereas in nonkeratinizing squamous cell carcinoma, they occur to a much lesser extent. Surprisingly, these keratins were also occasionally found in adenocarcinomas. From these data we conclude that the keratin phenotype of reserve cells and endocervical columnar cells is more complex than previously suggested. In particular, the keratins occurring in reserve cells are also present in most of the premalignant and in a considerable number of the malignant lesions of the cervix. The differentiation features of the various carcinoma types are, however, reflected in their specific keratin filament composition.

Among the members of the cytokeratin (CK) subfamily of intermediate filament (IF) proteins, CK 17 is remarkable as it is normally expressed in the basal cells of complex epithelia but not in stratified or simple epithelia. Because of its unusual expression pattern in normal and diseased states and because of the potential importance of CK 17 in tumor diagnosis, we have characterized the gene(s) and its cDNA-derived amino acid sequence. A cDNA clone encoding CK 17 was isolated from a HeLa cDNA library and used for the determination of the amino acid sequence, for studies of expression and for the screening of human genomic libraries. A number of lambda phage clones were isolated that covered three distinct, non-contiguous gene regions. Only one of these loci contains the functional CK 17 gene which is located only approximately 5 kbp 5'-upstream of the CK 16 gene, whereas the other two contain unprocessed CK 17 pseudogenes. Each of these genes is part of a larger CK type I gene locus the arrangement of which suggests that these genes and pseudogenes have arisen during evolution by duplication events comprising whole multigene loci. The functional CK 17 gene differs from the pseudogenes by the extent of methylation of certain DNA sequences in the 5'-upstream region. The 5 kbp CK 17 gene with 8 exons and 7 introns encodes a polypeptide of 432 amino acids with a calculated molecular weight of 48,000. Using S1-nuclease protection assays and RNAs from several cell lines we identified a single transcriptional start point 26 nucleotides down-stream from a TATA box element. Northern blot hybridization experiments showed a restricted pattern of CK 17 gene expression, supporting the notion that CK 17 synthesis is essentially regulated at the transcriptional level. From these findings and from immunohistological observations, CK 17 synthesis seems to be a marker of basal cell differentiation in complex epithelia and therefore indicative of a certain type of epithelial "stem cells".

In the present study the human cytokeratin 13 gene (KRT13), encoding a polypeptide characteristic of internal stratified epithelia, has been mapped with the help of the polymerase chain reaction and somatic cell hybrids to chromosome 17. In situ hybridization of a KRT13 cDNA probe to metaphase chromosomes allowed the assignment of the KRT13 gene within the q12-q21.2 region of chromosome 17.

Synaptophysin (SY) is an integral membrane protein of presynaptic small (30-80-nm) translucent vesicles also present in dispersed neuroendocrine cells. As the occurrence of this type of vesicle is specific for two major pathways of differentiation, the neuronal and neuroendocrine-epithelial information on the regulation of SY synthesis should contribute to an understanding of regulatory principles common to both pathways. Isolation and comparison of the complete rat and human single-copy genes showed that despite the difference in size (16 kb in rat vs. 13 kb in man) intron/exon boundaries are precisely conserved. Surprisingly, intron VI is located in the 3'-noncoding region in both species. The major transcriptional start point, as determined by primer extension and S1-nuclease protection analyses in rat pheochromocytoma-derived PC12 cells and rat brain, mapped to a site 27 nt 5' of the first methionine codon. Unexpectedly, the 5' upstream region is devoid of any TATA or CAAT boxes, but shows instead typical features of 'housekeeping' genes, i.e., G + C-rich islands and four Sp1-binding motifs. Using 'nuclear run-on' assays, we have identified examples in which SY synthesis is regulated at the transcriptional level. Reporter gene constructs showed that approx. 1.2 kb of the immediate upstream region contains promoter enhancer elements that were, however, insufficient to confer cell-type specific expression, whereas sequences farther upstream were able to suppress thymidine kinase promoter activity in a cell-type-dependent fashion.

Squamous cell metaplasia (SCM) is a frequent epithelial alteration of the human tracheobronchial mucosa. This review pays particular attention to the fact that SCM can mimic esophageal, and in some instances even skin-type differentiation, showing striking similarities not only in morphology but also in terms of gene expression. Therefore, characterization of this dynamic process lends insight into the process of stratification, squamous cell formation, and "keratinization" in a pathologically relevant in vivo situation in man. First, the concept of metaplasia is presented with certain historical viewpoints on histogenesis. Then, the morphological characteristics of normal bronchial epithelium are compared with the altered phenotype of cells in SCM. These changes are described as a disturbance of the finely tuned balance of differentiation and proliferation through the action of a variety of extrinsic and intrinsic factors. Molecular aspects of altered cell/cell and cell/extracellular matrix interactions in stratified compared with single-layered epithelia are discussed with reference to SCM in the lung. Intracellular organizational and compositional changes are then summarized with special emphasis on the differential distribution of the cytokeratin (CK) polypeptides. Finally, the still unresolved problems of the histogenetic relationships between normal bronchial mucosa, SCM, and pulmonary neoplasms are addressed. As these questions remain open, examples for detection of well defined "markers" are provided that may be employed as objective criteria for determining clinically important cellular differentiation features.

Small-cell carcinomas of the lung (SCLCs) are frequent tumors of the bronchopulmonary tract which are distinguished by their neuroendocrine (NE) features, indicative of a derivation from the sparse neuroendocrine cells present in the normal epithelium. Because of the lack of marked morphological details, the differential diagnosis of this tumor is very difficult. We show that the epithelial nature and origin of SCLCs can be demonstrated, biochemically and immunocytochemically, by the expression of cytokeratins, notably cytokeratins 8 and 18, often together with desmosomal proteins as another independent differentiation marker. The NE character of SCLCs, on the other hand, is revealed by antibodies to certain NE-specific components, notably the broad range NE marker, the membrane protein synaptophysin. Both kinds of differentiation markers are also expressed in a number of SCLC-derived cultured cell lines which therefore may serve as valuable biological model systems to study the biology of SCLC.

The chromosomal location of representative members of the type I and type II subfamilies of the cytokeratin multigene family was determined using specific cDNA probes in Southern blot hybridization with DNA from somatic cell hybrids. Our results show that the gene encoding human type II cytokeratin 4 resides on chromosome 12 and that encoding type I cytokeratin 15 is located on chromosome 17. The results indicate that cytokeratins are not concentrated in only one cluster. The possibility of the existence of separate type I and type II cytokeratin gene clusters is discussed.

We describe cDNA clones of mRNAs encoding human cytokeratins nos. 8 and 18, and the amino acid sequences deduced from their nucleotide sequences. Human cytokeratin no. 8 is a typical cytokeratin of the basic (type II) subfamily, which is highly homologous to the corresponding bovine and amphibian (Xenopus laevis) proteins; however, unlike the amphibian protein, it does not contain glycine-rich oligopeptide repeats in its carboxyterminal 'tail' domain. Comparison with the reported amino acid sequences of two fragments of human 'tissue polypeptide antigen' (TPA), a widely used serodiagnostic carcinoma marker, revealed sequence identity, indicating that this serum component is derived from the intracellular cytokeratin no. 8 present in diverse kinds of epithelia and epithelium-derived tumors. Human cytokeratin no. 18 is very similar to the corresponding murine protein but contains two additional blocks of 4 and 5 amino acids in the 'head' portion. These cDNA clones and the RNA probes derived therefrom were used to detect specifically mRNAs by Northern-blot assays of RNAs from various carcinomas and cultured carcinoma cells. Using in situ hybridization on frozen sections of tumor-containing tissues, notably lymph nodes containing metastatic breast carcinoma, we were able to demonstrate the specificity and sensitivity of this procedure. The potential value for cell-biological research and pathology of being able to detect a mRNA encoding a given cytokeratin polypeptide in situ is discussed.

Diverse nonneuroendocrine (non-NE) cells were forced to express synaptophysin (SY), the major and typical transmembrane glycoprotein of small (30-80 nm) neurotransmitter vesicles of NE cells, using microinjection of RNA synthesized in vitro from cDNA or transient and stable transfections with cDNA brought under SV40 promoter control. The glycoprotein synthesized in non-NE cells is indistinguishable from SY of NE cells and is integrated with similar, if not identical, orientation in the membranes of a specific, novel type of small cytoplasmic vesicle that structurally resembles synaptic vesicles and in which SY is the only major protein detected. A non-N-glycosylated form of SY generated by site-directed mutagenesis showed the same behavior and specific distribution in small vesicles. The results show that the information contained in this protein alone is sufficient to secure its sorting into a special type of vesicle in a heterotypic context, i.e., in the absence of other NE-specific components.

Human cytokeratin 13 is one of the most abundant intermediate filament (IF) proteins of many internal stratified epithelia and occurs, at least in certain cell cultures, in an O-glycosylated form binding the lectin, wheat germ agglutinin (WGA). As other groups have reported that, in the mouse, the synthesis of mRNA encoding the 47-kDa cytokeratin corresponding to human cytokeratin 13 is induced in epidermal keratinocytes during malignant transformation, we have examined the synthesis of cytokeratin 13 mRNA and protein in human epidermis and epidermal tumors, using specific cDNA probes and cytokeratin 13 antibodies. We isolated two different cDNA clones from the vulvar carcinoma cell line A-431, in which this protein is abundant: One clone seems to represent the entire mRNA, whereas the other is only a minor component and encodes a truncated cytokeratin 13 lacking most of the carboxy-terminal tail domain, probably a product of alternative, "incorrect" splicing. Comparison of the amino acid sequences with those of other cytokeratins revealed a high degree of conservation with respect to several other human type I cytokeratins, notably cytokeratin 15, and to the murine 47-kDa cytokeratin. When human epidermis and a series of benign and malignant epidermal tumors were examined with these cDNA probes and cytokeratin-13-specific antibodies we did not find an induction of expression in keratinocytes, normal or malignantly transformed, except for some scattered, sparse cytokeratin-13-positive cells and very low levels of cytokeratin 13 mRNA, detectable only with the highly sensitive polymerase chain reaction (PCR). We conclude that the gene(s) encoding cytokeratin 13 are not induced in human keratinocytes during epidermal carcinogenesis, in apparent contrast to reports of murine epidermal tumors, and we discuss possible explanations for this interspecies difference.