Epithelial growth and differentiation: an overview.
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Animals↗
Biomedical subjects
T T Sun
Publications and source records attributed to T T Sun.
Formation of asymmetric unit membrane during urothelial differentiation.
Mammalian urothelium undergoes unique membrane specialization during terminal differentiation making numerous rigid-looking membrane plaques (0.3-0.5 micron diameter) that cover the apical cell surface. The outer leaflet of these membrane plaques is almost twice as thick as the inner leaflet hence the name asymmetric unit membrane (AUM). Ultrastructural studies established that the outer leaflet of AUM is composed of 16 nm particles forming two dimensional crystals, and that each particle forms a 'twisted ribbon' structure. We showed recently that highly purified bovine AUMs contain four major integral membrane proteins: uroplakins Ia (27 kD), Ib (28 kD), II (15 kD) and III (47 kD). Studies of the protease sensitivity of the different subdomains of uroplakins and other considerations suggest that UPIa and UPIb have 4 transmembrane domains, while UPII and UPIII have only one transmembrane domain. Chemical crosslinking studies showed that UPIa and UPIb, which share 39% amino acid sequence, are topologically adjacent to UPII and UPIII, respectively, thus raising the possibility that there exist two biochemically distinct AUM particles, i.e., those containing UPIa/UPII vs. UPIb/UPIII. Bovine urothelial cells grown in the presence of 3T3 feeder cells undergo clonal growth forming stratified colonies capable of synthesizing and processing all known uroplakins. Transgenic mouse studies showed that a 3.6 kb 5'-flanking sequence of mouse uroplakin II gene can drive the expression of bacterial LacZ gene to express in the urothelium. Further studies on the biosynthesis, assembly and targeting of uroplakins will offer unique opportunities for better understanding the structure and function of AUM as well as the biology of mammalian urothelium.
Animals↗
Analysis of differentiation-associated proteins in rat bladder carcinogenesis.
Uroplakins are the major integral membrane proteins synthesized in terminally differentiated, superficial urothelial cells. Alteration of cell differentiation during rat urinary bladder carcinogenesis was analyzed immunohistochemically for the expression of uroplakins. Expression of uroplakins was compared in N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT)-, uracil-, sodium saccharin- or sodium ascorbate-induced urothelial simple hyperplasia, papillary-nodular hyperplasia, papilloma and carcinoma. In controls, uroplakins were located only in superficial cells, especially the luminal surface membrane. In FANFT-induced hyperplasia, including simple hyperplasia, intermediate cells also stained and the staining pattern was disorderly and intermittent. In uracil-induced simple hyperplasia, intermediate cells were stained but in an orderly fashion. In sodium saccharin- or sodium ascorbate-induced simple hyperplasia, superficial cells were swollen but alterations were not observed in the staining pattern. In carcinoma induced by FANFT and uracil, uroplakin expression was very disorderly and focal, usually with no expression on surface cells. It appears that disorderly differentiation is an index of bladder malignancy and is an early event in FANFT-induced lesions but a late event in uracil-, sodium saccharin- and sodium ascorbate-induced lesions.
Animals↗
Expression of keratohyalin-trichohyalin hybrid granules in molluscum contagiosum.
BACKGROUND: Recently, in the filiform papillae epithelium of mouse dorsal tongue, we showed the presence of hybrid granules in which filaggrin and trichohyalin were both present, but physically segregated. Further, trichohyalin was also detected in scattered granular cells of a number of hyperplastic skin diseases. METHODS: The epidermis infected with molluscum contagiosum virus (MCV) was studied by conventional electron microscopy in conjunction with light and electron-microscopic immunohistochemistry, using both antifilaggrin and antitrichohyalin antibodies as probes. RESULTS: We found that the granular cells of MCV-infected epidermis contained both filaggrin and trichohyalin. Subsequent electron-microscopic examination showed that the granular cells contained morphologically heterogeneous granules that appeared to be composed of discrete areas of distinct electron densities. Double-labeling, using antibodies to filaggrin and trichohyalin, clearly indicated that filaggrin and trichohyalin were both present in the hybrid granules and that the electron-dense regions contained trichohyalin while the more electron-lucent regions contained filaggrin. CONCLUSIONS: The expression of trichohyalin was a common feature observed in the epidermis from a heterogenous group of hyperplastic conditions, including MCV infection. This finding has led us to speculate that trichohyalin may be specifically or preferentially involved in interacting with the hyperproliferation-related keratin pair (K6/K16), whereas the function of filaggrin is more closely linked to the skin-type keratin pair (K1/K10) that are normal keratins found in the differentiated epidermis.
Cell Differentiation↗
Rabbit conjunctival and corneal epithelial cells belong to two separate lineages.
PURPOSE: This study investigated rabbit conjunctival and corneal epithelial cells to determine if they belong to two separate lineages. METHODS: Rabbit corneal, limbal, and conjunctival epithelial cells were isolated and grown in Dulbecco's minimum essential media and 20% fetal bovine serum in the presence of mitomycin-treated 3T3 feeder cells. After reaching 80% confluence, 3T3 feeder cells and any contaminating fibroblasts were removed, and epithelial cells were resuspended in fresh Dulbecco's minimum essential media. Aliquots containing 5x10(6) cells were placed subcutaneously into the flanks of athymic mice, which subsequently formed small nodules. At 2, 4, 6, 8, 14, 21, and 28 days, athymic mice were killed and the nodules (epithelial cyst) were excised for light and transmission electron microscopic examination and histochemical and cell kinetic analyses. RESULTS: Within 2 days after injection of single-cell suspensions, cells aggregated to form cysts lined with a stratified squamous epithelium, the structure of which resembled the original in vivo donor sites by 8 days. Limbal- and corneal-derived cysts were comprised only of glycogen-rich stratified epithelial cells. In contrast, only cysts arising from cultured conjunctival cells contained periodic acid-Schiff-positive cells with a goblet cell structure interspersed among stratified epithelial cells. Furthermore, cystic epithelium of conjunctival origin did not accumulate glycogen. CONCLUSIONS: To determine whether distinct phenotypes are caused by intrinsic divergence or by environmental modulation, the behavior of cells can be monitored in an identical in vivo growth environment. The athymic mouse provides such a permissive growth environment for cultured corneal, limbal, and conjunctival epithelial cells. All these cells reproduced their in vivo phenotype when placed in the athymic mouse. Thus, these findings provide the strongest evidence to date that the corneal-limbal lineage is distinct from the conjunctival lineage. These data also support the idea that the progenitor of goblet cells does not reside in the corneal-limbal epithelial compartment.
Animals↗
Selective interactions of UPIa and UPIb, two members of the transmembrane 4 superfamily, with distinct single transmembrane-domained proteins in differentiated urothelial cells.
The transmembrane 4 (TM4) superfamily contains many important leukocyte differentiation-related surface proteins including CD9, CD37, CD53, and CD81; tumor-associated antigens including CD63/ME491, CO-029, and SAS; and a newly identified metastasis suppressor gene R2. Relatively little is known, however, about the structure and aggregation state of these four transmembrane-domained proteins. The asymmetrical unit membrane (AUM), believed to play a major role in stabilizing the apical surface of mammalian urothelium thus preventing it from rupturing during bladder distention, contains two TM4 members, the uroplakins (UPs) Ia and Ib. In association with two other (single transmembrane-domained) membrane proteins, UPII and UPIII, UPIa and UPIb form 16-nm particles that naturally form two-dimensional crystalline arrays, thus providing unique opportunities for studying membrane structure and function. To better understand how these proteins interact to form the 16-nm particles, we analyzed their nearest neighbor relationship by chemical cross-linking. We show here that UPIa and UPIb, which share 39% of their amino acid sequence, are cross-linked to UPII and UPIII, respectively. We also show that UPIa has a propensity to oligomerize, forming complexes that are stable in SDS, and that UPII can be readily cross-linked to form homodimers. The formation of UPII homodimers is sensitive, however, to octyl glucoside that can solubilize the AUMs. These data suggest that there exist two types of 16-nm AUM particles that contain UPIa/UPII or UPIb/UPIII, and support a model in which the UPIa and UPII occupy the inner and outer domains, respectively, of the UPIa/UPII particle. This model can account for the apparent "redundancy" of the uroplakins, as the structurally related UPIa and UPIb, by interacting with different partners, may play different roles in AUM formation. The model also suggests that AUM plaques with different uroplakin compositions may differ in their assembly, and in their abilities to interact with an underlying cytoskeleton. Our data indicate that two closely related TM4 proteins, UPIa and UPIb, can be present in the same cell, interacting with distinct partners. AUM thus provides an excellent model system for studying the targeting, processing, and assembly of TM4 proteins.
Amino Acid Sequence↗
Ectopic expression of a bacterial lacZ gene in the limbic system of transgenic mice.
In three independent lines of transgenic mice, a 3.6 kb 5'-flanking sequence of the uroplakin II gene consistently drives the ectopic expression of a bacterial lacZ reporter gene in brain, in addition to its specific expression in the suprabasal layers of the urothelium. The ectopic expression in brain is especially noteworthy insofar as it is confined to structures comprising the limbic system. These findings provide additional evidence that the cells forming such functional systems share specific biochemical properties, and also indicate that this promoter may be useful as a tool for studying the effects of overexpression of proteins in anatomically and functionally defined central nervous system pathways.
Animals↗
Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed "twisted ribbon" structure.
The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rm) = 3.7 nm, outer ring radius (rout) = 6.6 nm), each with six domains which were rotated by roughly 30 degrees relative to each other. Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six "V-shaped" subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, "twisted ribbon" embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques.
Animals↗
A tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice.
Uroplakins are a group of integral membrane proteins that are synthesized as the major differentiation products of urothelium. The luminal portions of these proteins form 12-nm protein particles arranged in a two-dimensional crystalline array. The expression of uroplakin genes is bladder specific and differentiation dependent; little is known, however, about their molecular regulation. Here we describe the cloning of mouse uroplakin II gene and demonstrate, in transgenic mouse experiments, that a 3.6-kb 5'-flanking sequence of this gene can drive a bacterial lacZ (reporter) gene to express in the suprabasal cell layers of the urothelium. The transgene was not expressed in any tested (nonurothelial) epithelial and other tissues (except hypothalamus). These results suggest that most of the cis elements that confer the bladder-specific and differentiation-dependent expression of mouse uroplakin II gene must reside in the 3.6-kb sequence. The availability of a promoter capable of delivering a foreign molecule to the differentiated cell layers of bladder epithelium opens avenues for studying normal and pathological urothelial differentiation in transgenic mice.
Amino Acid Sequence↗
Hair follicle stem cells: present concepts.
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Animals↗
Characterization of hair follicle bulge in human fetal skin: the human fetal bulge is a pool of undifferentiated keratinocytes.
It has been suggested that the bulge of the hair follicle contains a pool of follicular stem cells that may serve as a target site of graft-versus-host disease and as a source of cells with carcinogenic potential. The bulge is prominent in the developing follicle although it is a subtle swelling in the adult follicle. In this paper, we studied the bulge in human fetal skin specimens. Ultrastructurally, the bulge cells, especially the interior cells, have abundant free ribosomes and glycogen particles, but almost no cytoplasmic organelles indicative of differentiation. Immunostaining with several specific anti-keratin antibodies demonstrated that the bulge cells express keratins of both stratified and simple epithelia. Melanocytes and Merkel cells, defined by immunohistochemical and ultrastructural criteria, are seen among bulge cells. Laser confocal microscopy revealed that primitive smooth muscle cells attached directly to the bulge initially at the mid-bulbous hair peg, the stage when the bulge is most prominent. K-laminin and type VII collagen are strongly expressed in the dermoepidermal junction of the bulge and between the matrix area of the bulb and the dermal papilla. Thus, the bulge of human hair follicle is not only an attachment site for arrector pili muscle, but also a pool of keratinocytes that are relatively undifferentiated.
Animals↗
Cutaneous ultrastructural features of the flaky skin (fsn) mouse mutation.
An autosomal recessive genetic disease with clinical and histopathological skin features resembling human psoriasis vulgaris occurs naturally in flaky skin mice (fsn/fsn). Affected mice are normal at birth, except for a hypochromic anemia. Subsequently, they develop hyperkeratotic plaques and acanthosis with elongation of rete ridges. Scanning electron microscopic examination revealed a greatly thickened epidermis, a sparsity of hairs and scale accumulations on the epidermal surface. Hair shafts had conspicuous pits, striations, and exophytic protrusions. Nails were bent at a 90 degrees angle with surface irregularities and accumulations of scale at the nail base. Transmission electron microscopic examination showed increased epidermal thickness, mitochondrial aberrations, and intraepidermal invasion by neutrophils. Keratohyalin abnormalities were detected using immunocytochemical staining for profilaggrin. At the dermal-epidermal junction, numerous macrophages and mast cells were seen in close proximity to focal dissolutions of the basement membrane. A high density of collagen fibers and cellular infiltrates were evident in the papillary dermis. This constellation of ultrastructural aberrations is typically found in psoriasis vulgaris and supports the theory that the flaky skin mouse mutation is a naturally occurring analog to one variety of human psoriasis vulgaris.
Anemia, Hypochromic↗
Message of nexin 1, a serine protease inhibitor, is accumulated in the follicular papilla during anagen of the hair cycle.
A group of specialized mesenchymal cells located at the root of the mammalian hair follicle, known as the follicular or dermal papillary cells, are involved in regulating the hair cycle, during which keratinocytes of the lower follicle undergo proliferation, degeneration and regrowth. Using the arbitrarily primed-PCR approach, we have identified a 1.3 kb messenger RNA that is present in large quantities in cultured rat follicular papillary cells, but not in skin fibroblasts. This mRNA encodes nexin 1, a potent protease inhibitor that can inactivate several growth-modulating serine proteases including thrombin, urokinase and tissue plasminogen activator. In situ hybridization showed that nexin 1 message is accumulated in the follicular papilla cells of anagen follicles, but is undetectable in keratinocytes or other skin mesenchymal cells. In addition, nexin 1 message level varies widely among several immortalized rat vibrissa papillary cell lines, and these levels correlate well with the reported abilities of these cell lines to support in vivo follicular reconstitution. These results suggest a possible role of nexin 1 in regulating hair follicular growth.
Amyloid beta-Protein Precursor↗
Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas.
Uroplakins (UPs) Ia, Ib, II, and III, transmembrane proteins constituting the asymmetrical unit membrane of urothelial umbrella cells, are the first specific urothelial differentiation markers described. We investigated the presence and localization patterns of UPs in various human carcinomas by applying immunohistochemistry (avidin-biotin-peroxidase complex method), using rabbit antibodies against UPs II and III, to paraffin sections. Positive reactions for UP III (sometimes very focal) were noted in 14 of the 16 papillary noninvasive transitional cell carcinomas (TCCs) (88%), 29 of the 55 invasive TCCs (53%), and 23 of the 35 TCC metastases (66%). Different localization patterns of UPs could be distinguished, including superficial membrane staining like that found in normal umbrella cells (in papillary carcinoma), luminal (microluminal) membrane staining (in papillary and invasive carcinoma), and, against expectations, peripheral membrane staining (in invasive carcinoma). Non-TCC carcinomas of various origins (n = 177) were consistently negative for UPs. The presence of UPs in metastatic TCCs represents a prime example of even advanced tumor progression being compatible with the (focal) expression of highly specialized differentiation repertoires. Although of only medium-grade sensitivity, UPs do seem to be highly specific urothelial lineage markers, thus operating up interesting histodiagnostic possibilities in cases of carcinoma metastases of uncertain origin.
Amino Acid Sequence↗
[Evidence that uroplakins exist in the intermediate cell of mouse bladder epithelium].
Asymmetric unit membrane (AUM) consisting of uroplakins is a remarkable structure in the apical plasma membrane of mammalian urothelium. The present study demonstrated that uroplakins existed in intermediate cells of mouse bladder epithelium and formed the typical AUM only in fusiform vesicles of the intermediate cells. The fusiform vesicle is also associated tightly with intermediate filament much like in those of superficial cells. This presents a reliable evidence for the morphogenesis of AUM and the differentiation model of bladder urothelium.
Animals↗
Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms.
BACKGROUND: The corneal epithelium converges at the peripheral zone (limbus) with the conjunctival epithelium, forming a continuous sheet with phenotypically distinct regions--central, limbal, and conjunctival. The epithelial basement membrane (EBM) is important for corneal functions and cell adhesion, but its regional composition is poorly understood. Current literature is controversial as to the occurrence of type IV collagen in the cornea. The aim of this study was to investigate in detail corneal basement membrane (BM) composition and correlate it with the differentiation state of contributing cells. EXPERIMENTAL DESIGN: Adult human corneas (N = 8) were cryosectioned and analyzed by immunofluorescence with antibodies to 15 BM components and to keratin 3, a marker of corneal epithelial differentiation. RESULTS: A novel type of spatial heterogeneity ("horizontal") in the EBM composition was found between the central cornea, limbus, and conjunctiva. Central EBM had type IV collagen alpha 3-alpha 5 chains, whereas limbal and conjunctival EBM contained alpha 1-alpha 2 chains and also laminin alpha 2 and beta 2 chains. Limbal EBM in addition had alpha 5(IV) chain. Laminin-1 (alpha 1 beta 1 gamma 1), laminin-5 (alpha 3 beta 3 gamma 2), perlecan, fibronectin, entactin/nidogen, and type VII collagen were seen in the entire EBM. Another novel type of BM heterogeneity ("vertical") was typical for the corneal Descemet's membrane: its stromal face had alpha 1(IV) and alpha 2(IV) chains and fibronectin, whereas alpha 3(IV)-alpha 5(IV) chains, entactin/nidogen, laminin-1, and perlecan were present on the endothelial face. CONCLUSIONS: Type IV collagen controversy is the result of the shifts of isoforms in the limbus and conjunctiva. These shifts and the appearance of additional laminins in the limbus may be related to the differentiation state of corneal cells contributing to the EBM formation. Novel types of BM heterogeneity in the human cornea are described: regional (horizontal) in the EBM and vertical in the Descemet's membrane. The first one may be a common feature of converging complex epithelia, whereas the second one may be another unique property of the Descemet's membrane.
Adult↗
Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis.
PURPOSE: To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS: The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in "S" phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS: These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis.
Animals↗
Functional importance of an Sp1- and an NFkB-related nuclear protein in a keratinocyte-specific promoter of rabbit K3 keratin gene.
We have shown previously that a 300-base pair (bp) 5' upstream sequence of rabbit keratin K3 gene (RK3) can function as a keratinocyte-specific promoter in transient transfection assays. Electrophoretic mobility shift assays using various overlapping and mutated oligonucleotides established that corneal keratinocyte nuclear proteins bound in vitro to two sites (B and E). Immunosupershift and UV cross-linking established that the keratinocyte nuclear binding protein of site B (5'-GGGGCTTTCC-3', -262 to -253 bp) was NFkB consisting of the p65 and p50 subunits. The E site contained an unusual GC-rich motif (5'-CCGCCCCCTG-3', -203 to -194 bp) whose sequence deviated from the Sp1 consensus in 4 out of 10 positions; this site bound an Sp 1-related keratinocyte nuclear protein. Mutagenesis of the NFkB, GC motif, and both sites abolished 20, 50, and 75%, respectively, of the promoter activity in transfected keratinocytes. The NFkB-like keratinocyte nuclear protein was barely detectable in kidney epithelial cells, HeLa, and fibroblasts. The Sp1-related nuclear protein was abundant in keratinocytes and simple epithelial cells, but was much less abundant in fibroblasts. These results indicate that NFkB is present in significant quantities in keratinocyte nuclei and that the tissue restriction of the NFkB- and Sp1-related proteins, in combination with other factors, may contribute to the keratinocyte specificity of RK3 promoter.
Animals↗