Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Anti-glomerular basement membrane glomerulonephritis: Characterization of an epitope, antibody response, and the potential role of IL-4Ralpha in disease progression

Anti-Glomerular Basement Membrane Glomerulonephritis (anti-GBM GM) is one of the earliest described autoimmune disorders. Patients present with proteinuria, anti-GBM antibodies, and renal failure. Studies have implicated a T Helper 1 (TH1) response in disease induction and a T Helper 2 (TH2) response for disease progression. A 13 amino acid long peptide sequence spanning residues 28 through 40 [pCol(28–40)] of the Collagen IV α3 non-collagen domain (Col IV α3 NCD) is immunogenic and induces anti-GBM GN. In order to fully understand disease initiation, this peptide was further characterized. Peptides were created containing one amino acid substitution for the entire length of pCol(28–40) and induction of anti-GBM GN was monitored. When residues 31, 33, or 34 contained the substitution, anti-GBM GN was unable to be induced. Thus, residues 31, 33, and 34 of pCol(28–40) are required for induction of anti-GBM. Glomerular injury is observed as early as 14 days post anti-GBM GN induction. However, the presence of anti-GBM antibodies is not observed until 20 days post immunization. An enlarged lymph node adjacent to the diseased kidney exhibits B cell activation after renal injury and produces antibodies toward GBM. Thus, anti-GBM antibodies are a consequence of the initial renal injury. Differences between disease susceptible and disease resistant rat strains exist in the expression of IL-4Rα, a major player in the TH2 response. IL-4Rα signaling is regulated by soluble IL-4Rα (sIL-4Rα). Low expression levels of sIL-4Rα result in the stabilization of IL-4 binding, while elevated expression sequesters IL-4. Quantitative PCR experiments noted low siL-4Rα expression levels in disease susceptible rats. Induction of an immune response toward sIL-4Rα in this strain was responsible for delayed disease progression in 15 out of the 17 experimental animals. Antibody transfer and in vivo biological activity experiments confirmed that delayed disease development was due to anti-sIL-4Rα antibodies. Together these experiments indicate that a T-cell epitope is required for activation of a TH1 autoimmune response and anti-GBM antibodies are a consequence of renal injury. More importantly, a role for IL-4Rα signaling is implicated in the progression of anti-GBM GN. ^

Reciprocal interactions between β1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: A different perspective in epithelial biology

Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of β1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4–2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a three-dimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of β1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant down-regulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Cross-modulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and β1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of β1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be “normalized” by manipulating either pathway.

Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle.

The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

Mouse Col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones.

We have isolated overlapping cDNAs encoding the N-terminal non-triple-helical region of mouse alpha 1(XVIII) collagen and shown that three different variants of alpha 1(XVIII) collagen exist. Each of the three variants shows characteristic tissue-specific expression patterns. Immunohistochemical studies show positive staining for alpha 1(XVIII) collagen along the basement membrane zones of vessels in the intestinal villi, the choroid plexus, skin, liver, and kidney. Thus, we conclude that alpha 1(XVIII) collagen may interact (directly or indirectly) with components in basement membrane zones or on the basal surface of endothelial/epithelial cells.

Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion

Reasons for performing study: The key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. For this to happen the structural and adhesion proteins of the basement membrane zone must be altered. Which proteins and how damage to them leads to the lamellar separation of laminitis is unknown. Objectives: To investigate lamellar hemidesmosome and cytoskeleton damage and basement membrane dysadhesion using light microscopy (LM) and immunofluorescence microscopy (IFM). Methods: Cryostat sections of lamellar tissues from 2 control and 6 Standardbred horses with oligofructose induced laminitis were studied using LM and IFM. Plectin, integrin alpha(6) and BP230 antibody was used to label hemidesmosome intracellular plaque proteins and anti-BP180 and anti-laminin 5 (L5) was used to label anchoring filament (AF) proteins. Cytoskeleton intermediate filaments were labelled using anti-cytokeratin 14. The primary antibodies of selected sections were double labelled to show protein co-localisation. Results: Laminitis caused reduction of transmembrane integrin alpha(6), the AF proteins BP180 and L5,and failure of co-localisation of BP180 and L5. Proteins of the inner hemidesmosomal plaque, plectin and BP230, were unaffected. Conclusions: Loss of co-localisation of L5 and BP180 suggests that, during the acute phase of laminitis, L5 is cleaved and therefore, the AFs connecting the epidermis to the dermis, fail. Without a full complement of AFs separation at the lamellar dermo-epidermal junction occurs. Potential relevance: Suppressing or inhibiting metalloproteinase activity may prevent L5 cleavage and therefore the lamellar dermo-epidermal separation of laminitis.

Alveolar haemorrhage in anti-glomerular basement membrane disease without detectable antibodies by conventional assays

Anti-glomerular basement membrane (anti-GBM) disease represents the spectrum of disease attributable to circulating anti-GBM antibodies. While active anti-GBM disease in the absence of circulating anti-GBM antibodies has been described, it is considered rare with the use of current routinely available assays. We report four subjects with features consistent with active anti-GBM antibody disease without detectable antibodies by routinely available enzyme linked immunosorbent assay (ELISA) and immunoblot techniques. All were smokers who presented with diffuse alveolar haemorrhage, minimal renal involvement, and undetectable anti-GBM antibodies. Seronegative anti-GBM disease with predominant pulmonary involvement may be more common than previously appreciated and should be part of the differential diagnosis for otherwise unexplained diffuse alveolar haemorrhage. Renal biopsy with immunofluorescent studies should be considered in the diagnostic evaluation of such subjects, including those with idiopathic pulmonary haemosiderosis.

Opening Basement Membrane Gaps During C. elegans Uterine-Vulval Attachment

The basement membrane (BM) is a highly conserved form of extracellular matrix that underlies or surrounds and supports most animal tissues. BMs are crossed by cells during various remodeling events in development, immune surveillance, or during cancer metastasis. Because BMs are dense and not easily penetrable, most of these cells must open a gap in order to facilitate their migration. The mechanisms by which cells execute these changes are poorly understood. A developmental event that requires the opening of a BM gap is C. elegans uterine-vulval connection. The anchor cell (AC), a specialized uterine cell, creates a de novo BM gap. Subsequent widening of the BM gap involves the underlying vulval precursor cells (VPCs) and the π cells, uterine neighbors of the AC through non-proteolytic BM sliding. Using forward and reverse genetic screening, transcriptome profiling, and live-cell imaging, I investigated how the cells in these tissues accomplish BM gap formation. In Chapter 2, I identify two potentially novel regulators of BM breaching, isolated through a large-scale forward genetic screen and characterize the invasion defect in these mutants. In Chapter 3, I describe single-cell transcriptome sequencing of the invasive AC. In Chapter 4, I describe the role of the π cells in opening the nascent BM gap. A complete developmental pathway for this process has been elucidated: the AC induces the π fate through Notch signaling, after which the π cells upregulate the Sec14 family protein CTG-1, which in turn restricts the trafficking of DGN-1 (dystroglycan), a laminin receptor, allowing the BM to slide. Chapter 5 outlines the implications of these discoveries.

Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.

Hepatocyte growth factor stimulates invasion across reconstituted basement membranes by a new human small intestinal cell line

Hepatocyte growth factor/scatter factor (HGF/SF) is a protein growth factor whose pleiotropic effects on epithelial cells include the stimulation of motility, mitosis and tubulogenesis. These responses are mediated by the cell surface tyrosine kinase receptor c-met. Because both the cytokine and receptor are found in the gastrointestinal tract, we have studied the effects of HGF/SF on transformed gut epithelial cells which express c-met. Here we describe the response of a new transformed human jejunal epithelioid cell line (HIE-7) to HGF/SF. Morphologically HIE-7 cells are immature. Their epithelial lineage was confirmed by reactivity with the epithelial specific antibodies AE1/AE3, Cam 5.2, Ber-EP4 and anti-EMA and is consistent with their expression of c-met mRNA and protein. In addition, electron microscopic analysis revealed the presence of primitive junctions and rudimentary microvilli, but features of polarization were absent. When grown on reconstituted basement membranes, HIE-7 cells formed closely associated multicellular cord-like structures adjacent to acellular spaces. However, the cells did not mature structurally, form lumen-like structures or express disaccharidase mRNA, even in the presence of recombinant HGF (rHGF). On the other hand, rHGF induced HIE-7 cells to scatter and stimulated their rapid migration in a modified wound assay. To determine whether the motogenic effect caused by rHGF is associated with HIE-7 cell invasiveness across reconstituted basement membranes, a Boyden chamber chemoinvasion assay was performed. rHGF stimulated a 10-fold increase in the number of HIE-7 cells that crossed the basement membrane barrier, while only stimulating a small increase in chemotaxis across a collagen IV matrix, suggesting that the cytokine activates matrix penetration by these cells. rHGF also stimulated the invasion of basement membranes by an undifferentiated rat intestinal cell line (IEC-6) and by two human colon cancer cell lines which are poorly differentiated (DLD-1 and SW 948). In contrast, two moderately well differentiated colon cancer cell lines (Caco-2 and HT-29) did not manifest an invasive response when exposed to rHGF. These results suggest that HGF/SF may play a significant role in the invasive behavior of anaplastic and poorly differentiated gut epithelial tumors.

Models for studying cellular invasion of basement membranes

Invasion of extracellular matrices is crucial to a number of physiological and pathophysiological states, including tumor cell metastasis, arthritis, embryo implantation, wound healing, and early development. To isolate invasion from the additional complexities of these scenarios a number of in vitro invasion assays have been developed over the years. Early studies employed intact tissues, like denuded amniotic membrane (1) or embryonic chick heart fragments (2), however recently, purified matrix components or complex matrix extracts have been used to provide more uniform and often more rapid analyses (for examples, see the following integrin studies). Of course, the more holistic view of invasion offered in the earlier assays is valuable and cannot be fully reproduced in these more rapid assays, but advantages of reproducibility among replicates, ease of preparation and analysis, and overall high throughput favor the newer assays. In this chapter, we will focus on providing detailed protocols for Matrigel-based assays (Matrigel=reconstituted basement membrane; reviewed in ref. (3)). Matrigel is an extract from the transplantable Engelbreth-Holm-Swarm murine sarcoma that deposits a multilammelar basement membrane. Matrigel is available commercially (Becton Dickinson, Bedford, MA), and can be manipulated as a liquid at 4°C into a variety of different formats. Alternatively, cell culture inserts precoated with Matrigel can be purchased for even greater simplicity.

Advanced glycation end product (AGE) accumulation on Bruch's membrane: links to age-related RPE dysfunction.

PURPOSE: Advanced glycation end products (AGEs) accumulate during aging and have been observed in postmortem eyes within the retinal pigment epithelium (RPE), Bruch's membrane, and subcellular deposits (drusen). AGEs have been associated with age-related dysfunction of the RPE-in particular with development and progression to age-related macular degeneration (AMD). In the present study the impact of AGEs at the RPE-Bruch's membrane interface was evaluated, to establish how these modifications may contribute to age-related disease. METHODS: AGEs on Bruch's membrane were evaluated using immunohistochemistry. A clinically relevant in vitro model of substrate AGE accumulation was established to mimic Bruch's membrane ageing. Responses of ARPE-19 growing on AGE-modified basement membrane (AGE-BM) for 1 month were investigated by using a microarray approach and validated by quantitative (q)RT-PCR. In addition to identified AGE-related mRNA alterations, lysosomal enzyme activity and lipofuscin accumulation were also studied in ARPE-19 grown on AGE-BM. RESULTS: Autofluorescent and glycolaldehyde-derived AGEs were observed in clinical specimens on Bruch's membrane and choroidal extracellular matrix. In vitro analysis identified a range of dysregulated mRNAs in ARPE-19 exposed to AGE-BM. Altered ARPE-19 degradative enzyme mRNA expression was observed on exposure to AGE-BM. AGE-BM caused a significant reduction in cathepsin-D activity in ARPE-19 (P

Differentiation status of limbal epithelial cells cultured on intact and denuded amniotic membrane before and after air-lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under ‘air-lifting’ conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting and quantitative PCR. Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane. On denuded amniotic membrane the proportion of differentiated cells remained unaltered following airlifting. Within cells grown on intact amniotic membrane, however, the number of differentiated cells increased significantly following air-lifting. These results have important implications for both basic and clinical research. Firstly, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Secondly, these findings serve as a warning to clinicians that the affect of amniotic membrane on transplantable cells is not fully understood; the use of intact or denuded amniotic membrane can produce different results in terms of the amount of differentiation, once cells are exposed to the air.