HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell–cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell–cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.     

Low Rho activity in hepatocytes prevents apical from basolateral cargo separation during trans‐Golgi network to surface transport

Hepatocytes, the main epithelial cells of the liver, organize their polarized membrane domains differently from ductal epithelia. They also differ in their biosynthetic delivery of single‐membrane‐spanning and glycophosphatidylinositol‐anchored proteins to the apical domain. While ductal epithelia target apical proteins to varying degrees from the trans‐Golgi network (TGN) to the apical surface directly, hepatocytes target them first to the basolateral domain, from where they undergo basolateral‐to‐apical transcytosis. How TGN‐to‐surface transport differs in both scenarios is unknown. Here, we report that the basolateral detour of a hepatocyte apical protein is due, in part, to low RhoA activity at the TGN, which prevents its segregation from basolateral transport carriers. Activating Rho in hepatocytic cells, which switches their polarity from hepatocytic to ductal, also led to apical‐basolateral cargo segregation at the TGN as is typical for ductal cells, affirming a central role for Rho‐signaling in different aspects of the hepatocytic polarity phenotype. Nevertheless, Rho‐induced cargo segregation was not sufficient to target the apical protein directly; thus, failure to recruit apical targeting machinery also contributes to its indirect itinerary.     

Transcriptome atlas of eight liver cell types uncovers effects of histidine catabolites on rat liver regeneration

Eight liver cell types were isolated using the methods of Percoll density gradient centrifugation and immunomagnetic beads to explore effects of histidine catabolites on rat liver regeneration. Rat Genome 230 2.0 Array was used to detect the expression profiles of genes associated with metabolism of histidine and its catabolites for the above-mentioned eight liver cell types, and bioinformatic and systems biology approaches were employed to analyse the relationship between above genes and rat liver regeneration. The results showed that the urocanic acid (UA) was degraded from histidine in Kupffer cells, acts on Kupffer cells itself and dendritic cells to generate immune suppression by autocrine and paracrine modes. Hepatocytes, biliary epithelia cells, oval cells and dendritic cells can convert histidine to histamine, which can promote sinusoidal endothelial cells proliferation by GsM pathway, and promote the proliferation of hepatocytes and biliary epithelia cells by GqM pathway.     

Par1b Induces Asymmetric Inheritance of Plasma Membrane Domains via LGN-Dependent Mitotic Spindle Orientation in Proliferating Hepatocytes

The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical plasma membrane domain to the nascent daughter cells. The non-polarized nascent daughter cell can form a de novo apical domain with its new neighbor. This asymmetric segregation of apical domains is facilitated by a geometrically distinct “apicolateral” subdomain of the lateral surface present in hepatocytes. The polarity protein partitioning-defective 1/microtubule-affinity regulating kinase 2 (Par1b/MARK2) translates this positional landmark to cortical polarity by promoting the apicolateral accumulation of Leu-Gly-Asn repeat-enriched protein (LGN) and the capture of nuclear mitotic apparatus protein (NuMA)–positive astral microtubules to orientate the mitotic spindle. Proliferating hepatocytes thus display an asymmetric inheritance of their apical domains via a mechanism that involves Par1b and LGN, which we postulate serves the unique tissue architecture of the developing liver parenchyma.     

Liver stem cells: a two compartment system

Hepatocytes and biliary epithelia are phenotypically very dissimilar, but share a common ancestry. Hepatocytes regenerate very efficiently, and their division potential indicates that many of them are functional stem cells. When hepatocyte-damaging agents also impair the regenerative ability of surviving hepatocytes, a potential stem cell system of biliary origin is activated to generate new hepatocytes — a reversal of ontogeny. Now both bile duct derived cells and hepatocytes can be isolated from the liver, genetically modified in vitro and returned to their in vivo origins where, after considerable population expansion, they can function as hepatocytes — paving the way for ex vivo gene therapy.     

Prolyl 4-hydroxylase isoenzymes I and II have different expression patterns in several human tissues.

Prolyl 4-hydroxylase plays a central role in the synthesis of all collagens. We have previously reported that the recently identified Type II isoenzyme is its main form in chondrocytes and possibly in capillary endothelial cells, while Type I is the main form in many other cell types. We report here that the Type II isoenzyme is clearly the main form in capillary endothelial cells and also in cultured umbilical vein endothelial cells, whereas no Type I isoenzyme could be detected in these cells by immunostaining or Western blotting. The Type II isoenzyme was also the main form in cells of the developing glomeruli in the fetal kidney and tubular structures of collecting duct caliber in both fetal and adult kidney, in occasional sinusoidal structures and epithelia of the bile ducts in the liver, and in some cells of the decidual membrane that probably represented invasive cytotrophoblasts in the placenta. Osteoblasts in a fetal calvaria, i.e., a bone developing by intramembranous ossification, stained strongly for both types of isoenzyme. The Type I isoenzyme was the main form in undifferentiated interstitial mesenchymal cells of the developing kidney, for example, and in fibroblasts and fibroblastic cells in many tissues. Skeletal myocytes and smooth muscle cells appeared to have the Type I isoenzyme as their only prolyl 4-hydroxylase form. Hepatocytes expressed small amounts of the Type I enzyme and very little if any Type II, the Type I expression being increased in malignant hepatocytes and cultured hepatoblastoma cells. The data suggest that the Type I isoenzyme is expressed especially by cells of mesenchymal origin and in developing and malignant tissues, whereas the Type II isoenzyme is expressed, in addition to chondrocytes and osteoblasts, by more differentiated cells, such as endothelial cells and cells of epithelial structures. (J Histochem Cytochem 49:1143-1153, 2001)     

Polarized signaling: basolateral receptor localization in epithelial cells by PDZ-containing proteins

Extracellular signals are normally presented to one surface of epithelial cells and to one end of neurons, and so neuronal and epithelial cell signaling is inherently polarized. Another aspect of signaling polarity is that receptors are often asymmetrically distributed on the surfaces of polarized cells. Recent evidence from studies of Caenorhabditis elegans shows that signaling polarity plays an important role in development. The underlying mesoderm induces the overlying ectoderm to form the vulva, and asymmetric distribution of the signal receptor on the basolateral surface of the epithelium is crucial for this signaling. In neurons, the localization of neurotransmitter receptors and ion channels at synapses allows neurons to be exquisitely sensitive to synaptic inputs. Exciting recent reports suggest that receptor localization to neuronal synapses and the basolateral membrane domains of epithelia may involve a common molecular mechanism involving localization by PDZ-containing proteins.     

Interaction of different types of epithelium in a mixed culture

Interaction of several lines of epithelial cells was studied in a mixed culture: FBT (bovine fetal trachea), MDSK (dog kidney), IAR-2 (rat liver), MPTR (SV-40 transformed mouse kidney). During mixed cultivation epithelia cells of different types were capable to force out each other from the substrate to full elimination. This capacity correlated with the pattern of cell contacts with the substrate. It is supposed that epithelial cells can form lamellae at the lower surfaces competing for substrate. Those cells which have lamellae with continuous marginal focal contacts and numerous focal contacts eliminate the cells having lamellae with few focal contacts.     

Daunorubicin-induced DNA lesions in isolated rat hepatocytes and mammary epithelial cells

Daunorubicin, an anticancer drug, induces primarily mammary adenocarcinoma in Sprague-Dawley rats. We investigated daunorubicin-induced DNA lesions in enzymatically isolated mammary epithelial cells and hepatocytes from 7-8-week-old female Sprague-Dawley rats. Differences were observed in the type and quantity of DNA lesions in mammary epithelial cells and hepatocytes as determined by alkaline elution analysis. DNA single-strand breaks and proteinase-K-sensitive cross-linking lesions were observed in mammary epithelial cells. Hepatocytes appeared to have significantly lower relative frequencies of single-strand breaks than mammary epithelial cells when treated with daunorubicin (1.5-10.0 micrograms/10(6) cells). Hepatocytes displayed two types of cross-link. One form was sensitive to proteinase-K digestion, whereas the other form was insensitive. The metabolism of daunorubicin to the aglycone metabolites was substantially lower in mammary cells than in hepatocytes. However, the total uptake of the drug was similar in these two cell types. A metabolite, 7-deoxydaunorubicinol aglycone, was unable to induce single-strand breaks or cross-linking lesions in mammary epithelial cells. Both cell types exhibited a similar ability to repair radiation-induced single-strand breaks of DNA. However, the mammary cells may be less able to repair daunorubicin-mediated DNA damage. These results revealed that mammary epithelial cells are less able to metabolize the active mutagen/carcinogen, daunorubicin, than are hepatocytes. This, coupled with the observations of greater apparent DNA damage in mammary cells, may be of primary importance in the drug-induced carcinogenicity in the rat mammary tissue.     

Epithelium--the primary building block for metazoan complexity

In simplest terms, the complexity of the metazoan body arisesthrough various combinations of but two tissue types: epitheliumand mesenchyme. Through mutual inductions and interactions,these tissues produce all of the organs of the body. Of thetwo, epithelium must be considered the default type in the Eumetazoabecause it arises first in embryonic development and becausemesenchyme arises from it by a switching off of the mechanismsthat underly differentiation and maintenance of epithelial cells.In the few model metazoans whose epithelia have been studiedby molecular techniques (largely Drosophila, Caenorhabditis,mouse), the molecular mechanisms underlying differentiationof epithelia show remarkable similarity. Extrapolating fromthese studies and from comparisons of the morphology of epitheliain lower metazoans, I propose how epithelia arose in the stemmetazoan. Steps in epithelial differentiation include 1) establishmentof cell polarity by molecular markers confined to either apicalor basolateral domains in the plasma membrane; 2) aggregationof cells into sheets by localization of cell-adhesion moleculeslike cadherin to the lateral membrane; 3) formation of a zonulaadherens junction from the cadherins by their localization toa discrete belt; 4) cell-to-cell linking of certain transmembraneproteins (primitively in the septate junction) to produce gatesthat physiologically isolate compartments delimited by the cells;and 5) synthesis of a basal lamina and adaptation of receptors(integrins) to its components. Despite morphological differencesin the variety of cell junctions evident in various epithelia,the underlying molecular markers of these junctions are probablyuniversally present in all eumetazoan epithelia.     

Isolation and characterization of biliary epithelial cells from rainbow trout liver

Summary Lectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment to coverslips coated withPhaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their response to toxic and carcinogenic agents.     

Septate junctions in imaginal disks of Drosophila: a model for the redistribution of septa during cell rearrangement

The organization of septate junctions during morphogenesis of imaginal disks is described from freeze-fracture replicas and thin sections with a view to understanding junction modulation during rearrangements of cells in epithelia. The septate junctions of each epithelial cell of the disk are distributed in a number of discrete domains equal to the number of neighboring cells. Individual septa traverse domains of contact between pairs of adjacent cells, turn downwards at the lateral boundary of the domain and run parallel to the intersection with a third cell. This arrangement leaves small channels at three-cell intersections that are occupied by specialized structures termed "tricellular plugs." Cell rearrangement involves a progressive change in the width of contact domains between adjacent cells, until old contacts are broken and new ones established. It is proposed that the septate junction adjusts to the changing width of domains by the compaction or extension of existing septa. This redistribution of septa theoretically allows a transepithelial barrier to be maintained during cell rearrangements. The applicability of this model to other epithelial tissues is discussed.     

Vectorial TGFβ signaling in polarized intestinal epithelial cells

Polarized gastrointestinal epithelial cells form tight junctions that spatially separate apical and basolateral cell membrane domains. These domains harbor functionally distinct proteins that contribute to cellular homeostasis and morphogenesis. Transforming growth factor β (TGFβ) is a critical regulator of gastrointestinal epithelial cell growth and differentiation. Functional assays of vectorial TGFβ signaling and immunofluorescence techniques were used to determine the localization of TGFβ receptors and ligand secretion in polarizing Caco‐2 cells, a colon cancer cell line. Results were compared to the nontransformed MDCK cell line. In both Caco‐2 and MDCK cells, addition of TGFβ1 to the basolateral medium resulted in phosphorylation of Smad2. No phosphorylation was observed when TGFβ1 was added to the apical chamber, indicating that receptor signaling is localized at the basolateral membrane. In support of this, immunofluorescence and biotinylation assays show receptor localization along the basolateral membrane. Secretion of TGFβ1 from MDCK and Caco‐2 cells into the apical or basolateral medium was measured by ELISA. Interestingly, secretion was exclusively apical in the nontransformed MDCK line and basolateral in transformed Caco‐2 cells. Collectively, these results show basolateral domain specificity in localization of the TGFβ receptor signaling apparatus. These observations have important implications for understanding the biology of TGFβ in polarized epithelia, including elements of communication between epithelial and mesenchymal layers, and will prove useful in the design of therapeutics that target TGFβ function. J. Cell. Physiol. 224: 398–404, 2010. © 2010 Wiley‐Liss, Inc.     

Epiplakin is dispensable for skin barrier function and for integrity of keratin network cytoarchitecture in simple and stratified epithelia

Epiplakin, a giant epithelial protein of >700 kDa, belongs to the plakin family of cytolinker proteins. It represents an atypical family member, however, as it consists entirely of plakin repeat domains but lacks any of the other domains commonly shared by plakins. Hence, its putative function as a cytolinker protein remains to be shown. To investigate epiplakin's biological role, we generated epiplakin-deficient mice by gene targeting in embryonic stem cells. Epiplakin-deficient mice were viable and fertile, without developing any discernible phenotype. Ultrastructurally, their epidermis revealed no differences compared to wild-type littermates, and cornified envelopes isolated from skin showed no alterations in shape or stability. Furthermore, neither embryonal formation nor later function of the epithelial barrier was affected. In primary cultures of epiplakin-deficient keratinocytes, the organization of actin filaments, microtubules, and keratin networks was found to be normal. Similarly, no alterations in keratin network organization were observed in simple epithelia of small intestine and liver or in primary hepatocytes. We conclude that, despite epiplakin's abundant and highly specific expression in stratified and simple epithelia, its absence in mice does not lead to severe skin dysfunctions, nor has it detectable consequences for keratin filament organization and cytoarchitecture of cells.     

Syndecan, a developmentally regulated cell surface proteoglycan that binds extracellular matrix and growth factors

Cellular behaviour during development is dictated, in part, by the insoluble extracellular matrix and the soluble growth factor peptides, the major molecules responsible for integrating cells into morphologically and functionally defined groups. These extracellular molecules influence cellular behaviour by binding at the cell surface to specific receptors that transduce intracellular signals in various ways not yet fully clear. Syndecan, a cell surface proteoglycan found predominantly on epithelia in mature tissues binds both extracellular matrix components (fibronectin, collagens I, III, V, and thrombospondin) and basic fibroblast growth factor (bFGF). Syndecan consists of chondroitin sulfate and heparan sulphate chains linked to a 31 kilodalton (kDa) integral membrane protein. Syndecan represents a family of integral membrane proteoglycans that differ in extracellular domains, but share cytoplasmic domains. Syndecan behaves as a matrix receptor: it binds selectively to components of the extracellular matrix, associates intracellularly with the actin cytoskeleton when cross-linked at the cell surface, its extracellular domain is shed upon cell rounding and it localizes solely to basolateral surfaces of simple epithelia. Mammary epithelial cells made syndecan-deficient become fibroblastic in morphology and cell behaviour, showing that syndecan maintains epithelial cell morphology. Syndecan changes in quantity, location and structure during development: it appears initially on four-cell embryos (prior to its known matrix ligands), becomes restricted in the pre-implementation embryo to the cells that will form the embryo proper, changes its expression due to epithelial-mesenchymal interactions (for example, induced in kidney mesenchyme by the ureteric bud), and with association of cells with extracellular matrix (for example, during B-cell differentiation), and ultimately, in mature tissues becomes restricted to epithelial tissues. The number and size of its glycosaminoglycan chains vary with changes in cell shape and organization yielding tissue type-specific polymorphic forms of syndecan. Its interactions with the major extracellular effector molecules that influence cell behaviour, its role in maintaining cell shape and its spatial and temporal changes in expression during development indicate that syndecan is involved in morphogenesis.     

Conserved form and function of the germinal epithelium through 500 million years of vertebrate evolution

The germinal epithelium, i.e., the site of germ cell production in males and females, has maintained a constant form and function throughout 500 million years of vertebrate evolution. The distinguishing characteristic of germinal epithelia among all vertebrates, males, and females, is the presence of germ cells among somatic epithelial cells. The somatic epithelial cells, Sertoli cells in males or follicle (granulosa) cells in females, encompass and isolate germ cells. Morphology of all vertebrate germinal epithelia conforms to the standard definition of an epithelium: epithelial cells are interconnected, border a body surface or lumen, are avascular and are supported by a basement membrane. Variation in morphology of gonads, which develop from the germinal epithelium, is correlated with the evolution of reproductive modes. In hagfishes, lampreys, and elasmobranchs, the germinal epithelia of males produce spermatocysts. A major rearrangement of testis morphology diagnoses osteichthyans: the spermatocysts are arranged in tubules or lobules. In protogynous (female to male) sex reversal in teleost fishes, female germinal epithelial cells (prefollicle cells) and oogonia transform into the first male somatic cells (Sertoli cells) and spermatogonia in the developing testis lobules. This common origin of cell types from the germinal epithelium in fishes with protogynous sex reversal supports the homology of Sertoli cells and follicle cells. Spermatogenesis in amphibians develops within spermatocysts in testis lobules. In amniotes vertebrates, the testis is composed of seminiferous tubules wherein spermatogenesis occurs radially. Emerging research indicates that some mammals do not have lifetime determinate fecundity. The fact emerged that germinal epithelia occur in the gonads of all vertebrates examined herein of both sexes and has the same form and function across all vertebrate taxa. Continued study of the form and function of the germinal epithelium in vertebrates will increasingly clarify our understanding of vertebrate reproduction. J. Morphol. 277:1014–1044, 2016. © 2016 Wiley Periodicals, Inc.     

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell–cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.     

Regeneration of the intestinal epithelia: Regulation of bone marrow-derived epithelial cell differentiation towards secretory lineage cells

The intestinal epithelia consists of four lineages of differentiated cells, all of which arise from stem cells residing in the intestinal crypt. For proper regeneration from epithelial damage, both expansion of the epithelial cell number and appropriate regulation of lineage differentiation from the remaining stem cells are thought to be required. In a series of studies, we have shown that bone-marrow derived cells could promote the regeneration of damaged epithelia in the human intestinal tract. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract of bone-marrow transplant recipients during epithelial regeneration after graft-versus-host disease. Furthermore, precise analysis of epithelial cell lineages revealed that during epithelial regeneration, secretory lineage epithelial cells that originated from bone-marrow significantly increased in number. These findings may lead to a novel therapy to repair damaged intestinal epithelia using bone marrow cells, and provide an alternative therapy for refractory inflammatory bowel diseases.