Faculty Bibliography

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

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

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

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

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

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

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

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