cDNA and genomic cloning of lacritin, a novel secretion enhancing factor from the human lacrimal gland.

Multiple extracellular factors are hypothesized to promote the differentiation of unstimulated and/or stimulated secretory pathways in exocrine secretory cells, but the identity of differentiation factors, particularly those organ-specific, remain largely unknown. Here, we report on the identification of a novel secreted glycoprotein, lacritin, that enhances exocrine secretion in overnight cultures of lacrimal acinar cells which otherwise display loss of secretory function. Lacritin mRNA and protein are highly expressed in human lacrimal gland, moderately in major and minor salivary glands and slightly in thyroid. No lacritin message or protein is detected elsewhere among more than 50 human tissues examined. Lacritin displays partial similarity to the glycosaminoglycan-binding region of brain-specific neuroglycan C (32 % identity over 102 amino acid residues) and to the possibly mucin-like amino globular region of fibulin-2 (30 % identity over 81 amino acid residues), and localizes primarily to secretory granules and secretory fluid. The lacritin gene consists of five exons, displays no alternative splicing and maps to 12q13. Recombinant lacritin augments unstimulated but not stimulated acinar cell secretion, promotes ductal cell proliferation, and stimulates signaling through tyrosine phosphorylation and release of calcium. It binds collagen IV, laminin-1, entactin/nidogen-1, fibronectin and vitronectin, but not collagen I, heparin or EGF. As an autocrine/paracrine enhancer of the lacrimal constitutive secretory pathway, ductal cell mitogen and stimulator of corneal epithelial cells, lacritin may play a key role in the function of the lacrimal gland-corneal axis.

Role of the basement membrane in tumor cell dormancy and cytotoxic resistance.

OBJECTIVES AND METHODS: Tumor dormancy and resistance to cytotoxic agents are key limiting events in the treatment of malignant diseases. To determine whether both are influenced by the extracellular milieu in which tumors reside, HT1080 human fibrosarcoma, MCF-7 breast carcinoma and OSCORT osteosarcoma cell proliferation, viability, apoptosis and cytoreductive-treatment-induced death were investigated in the presence or absence of extracellular matrix (ECM). RESULTS: ECM-adherent, but not plastic-adherent HT1080 cells formed a multicellular network accompanied by reduced proliferation and lowered DNA synthetic capacity. The number of cells in S-phase was dramatically reduced. Viable cells entered a state of dormancy reminiscent of that observed in the step of metastasis after extravasation, i.e. prior to the initiation of progressive growth. Such ECM-induced dormancy could be reversed by plating cells on plastic, but only after a 48-hour lag period. No difference was indicated in clonogenicity of HT1080 cells originated from plastic or ECM gel. However, the cells released from ECM gel showed significantly reduced migration ability. The resistance of anchored cells against cytotoxic damage was increased by ECM gel. Examination of cytoreductive treatment revealed that ECM adherence at the time of injury is partially protective, a property which was also moderately apparent when injured cells were transferred to the basement membrane. CONCLUSIONS: Taken together, these results suggest that the ECM plays a key role in tumor dormancy and cytotoxic resistance, both explorable at the molecular level using our in vitro model system.

Integrin expression and usage by prostate cancer cell lines on laminin substrata.

During prostate cancer progression, invasive glandular epithelial cells move out of the ductal-acinar architecture and through the surrounding basement membrane. Extracellular matrix proteins and associated soluble factors in the basal lamina and underlying stroma are known to be important regulators of prostate cell behaviors in both normal and malignant tissues. In this study, we assessed cell interactions with extracellular matrix and stromal factors during disease progression by characterizing integrin usage and expression in a series of parental and lineage-derived LNCaP human prostate cancer cell lines. Although few shifts in integrin expression were found to accompany disease progression, integrin heterodimer usage did change significantly. The more metastatic sublines were distinct in their use of alphavbeta3 and, when compared with parental LNCaP cells, showed a shift in alpha6 heterodimerization, a subunit critical not only for interaction with prostate basal lamina but also for interaction with the bone matrix, a favored site of prostate cancer metastases.

Human genome search in celiac disease using gliadin cDNA as probe.

Celiac disease is a wheat gliadin-promoted disorder that displays a complex genetic susceptibility associated with HLA-DQ2, and one or more unknown factor(s), possibly gliadin-like. The presence of mammalian proteins with partial gliadin similarity was suggested by transglutaminase-independent multi-tissue reactivity of gliadin-immunopurified antibodies from celiac patients. No non-plant sequence, however, was identified in gliadin peptide epitope searches of non-redundant and EST databanks via TBLASTN, BLASTP and FASTA, even at E values as high as 20. Therefore, an alpha-gliadin cDNA screen of human cDNA and genomic libraries was undertaken, an approach in keeping with positive human Northern and Southern analyses with the same probe. Four distinct cDNA clones were obtained, the most stringent of which (3.2 and 5.1 kb) were novel, and featured potential open reading frames with high gliadin domain II and domain IV homologies (BestFit quality scores >/=295 and 322, respectively, versus random value 126-127). Both were also homologous to ESTs. An additional 5' gliadin oligonucleotide screen identified the widely distributed cytoplasmic protein acyl coA hydrolase whose homology was restricted to the oligonucleotide probe (BestFit quality=215 versus 100 for random); and achaete-scute homologous protein, which displays particularly high gliadin domain II homology (BestFit quality 316 versus 111 for random). Genomic screening uncovered 16 positives, one of which was the ALR gene, whose similarity to three of gliadin's five domains (I, II and IV; BestFit quality 322-473 versus 121-154 for random) was remarkable. More extensive was novel genomic clone 2, with fragments hybridizing to cDNA probes approximating gliadin domains I, II+IV, V and the gliadin 5' untranslated region, and mapping by FISH to 19q13.11-13. 12. Two fragments were sequenced; one was exonic, as predicted by four different programs; and test oligonucleotides suggested widespread 4 and/or 2 kb mRNA expression, even at high stringency (t(m)-8.8 deg. C). Taken together, it is apparent that several genes with partial gliadin homology exist in the human genome. Many bear gliadin-like T-cell epitopes, are expressed in intestine and, like transglutaminase, are cytoplasmic. Glutamine to glutamic acid or other mutation within such epitopes followed by injury or infection-related release could explain enhanced disease susceptibility in affected families.

Quantitation of rat lacrimal secretion: a novel sandwich ELISA with high sensitivity.

Modulation of lacrimal acinar cell tear secretion may involve multiple factors acting both in subtle and pronounced ways. Functional screens of recombinant protein products arising from gene array technologies, or protein fractions derived from lacrimal conditioned media or extracellular matrix, will require a highly sensitive assay capable of monitoring tear protein secretion by small replicate cultures. To improve significantly on current methods, a rat- and mouse-specific sandwich ELISA was developed. For this purpose, chickens and rabbits were immunized with serum-free secretion media from carbachol and VIP-stimulated rat lacrimal acinar cell cultures. Immune sera were characterized by ELISA, Western blotting and immunohistochemistry, and subsequently optimized for use in a sandwich ELISA. Both antisera detected a wide range of different rat tear proteins, and immunostained only the secretory granule-rich juxtalumenal region in sections of rat lacrimal gland. Chicken, but not rabbit, antiserum cross-reacted with rabbit and human tears. In sandwich ELISA, capture with purified chicken immunoglobulin fraction and detection with rabbit antiserum detected as little as 1 ng ml-1 tear protein in 10,000-fold diluted rat secretion medium--a level of sensitivity 8000 times greater than the rat tear peroxidase assay. Such specificity and sensitivity greatly reduce the quantity of media needed for assay, and makes feasible functional screens for scarce factors that may influence lacrimal secretory processes, and in turn possibly play a role in human lacrimal insufficiency syndromes.

Role of laminin-1, collagen IV, and an autocrine factor(s) in regulated secretion by lacrimal acinar cells.

Adhesion to novel basement membrane component BM180 in the presence of laminin-1 promotes stimulus-secretion coupling in lacrimal acinar cells [G. W. Laurie, J. D. Glass, R. A. Ogle, C. M. Stone, J. R. Sluss, and L. Chen. Am. J. Physiol. 270 (Cell Physiol. 39): C1743-C1750, 1996]. The identity of the active laminin-1 site and the possibility that other promoters of coupling are present in the acinar cell microenvironment were probed by use of different substrates, media, neutralizing antibodies and cell numbers. Regulated peroxidase secretion was unaffected by basement membrane coat concentration and was detectable at reduced levels in serum-free medium. Anti-laminin-1 antibodies, particularly against sites in the beta1 and gamma1 chains, but not alpha1 chains, partially suppressed regulated secretion, as did an anti-collagen IV antibody. Without effect were RGD peptide and antibodies against entactin, the beta1-integrin subunit, and several growth factors. Increasing cell number in serum-free medium revealed an unknown, serum-maskable, secretion-enhancing activity with a remarkable specificity for regulated secretion. Stimulus-secretion coupling, therefore, appears to be modulated by several extracellular factors whose relative contributions remain to be determined.

Gene cloning of BM180, a lacrimal gland enriched basement membrane protein with a role in stimulated secretion.

BM180 is a novel basement membrane component with a role in regulated tear secretion by lacrimal acinar cells. BM180 bears some sequence similarity to alpha-gliadin, a plant protein against which antibodies have been reported in patients with Sjögren's syndrome. A precedent for plantlike sequence in the mammalian genome is provided by selectins, which possess a plant lectinlike domain involved in inflammatory cell homing. Cloning the mouse and human BM180 gene will aid molecular investigation of lacrimal acinar cell-BM180 interactions and may lead to a new molecular understanding of mechanisms contributing to dry eye.

Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading.

Cell adhesion to amino acids 2179-2198 (SN-peptide) of the laminin-1 alpha1-chain is required for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The nature of the SN-peptide receptor(s) was probed with neutralizing anti-integrin monoclonal antibodies (MAb), cells lacking integrin subunits, soluble heparin, and SN-peptide columns. Cell adhesion and spreading studies confirmed the specificity of SN-peptide and revealed adhesion to be unaffected by inclusion of anti-beta1-, anti-alpha(2-6)- or anti-alpha(V)beta5-integrin MAb. Cells lacking beta1- or alpha6-integrin subunits were fully adherent. Adhesion was heparin, but not chondroitin sulfate or heparinase, sensitive, much as is alpha-dystroglycan-laminin-1 binding. Heparin eluted approximately 155- and 180-kDa cell-surface proteins from SN-peptide columns. An additional approximately 91-kDa protein was eluted by EDTA. All were unrecognized by anti-beta1-integrin MAb. SN-peptide therefore interacts with three cell-surface proteins for which the identity remains to be determined.

Characterization of a basement membrane protein (BM180) using capillary electrophoresis.

Coeliac disease (gluten intolerance) is a genetically based autoimmune disease that becomes evident following ingestion of cereal prolamins such as the wheat gliadins. The process of this disease is not yet thoroughly understood. Purification of basement membrane protein-180 (BM180) (a basement membrane protein with a potential autoantigen role) and multiple analysis of the purified protein can lead to a better understanding of this disorder, which affects millions of people worldwide. Our preliminary work on the purification and characterization of this protein is presented in this paper. BM180 proteins were expressed in mouse EHS (Engelberth-Holm-Swarm) tumor cells. A crude purification process (see "Methods") was performed and the purified fractions were analyzed by capillary gel electrophoresis (CGE) for determination of the apparent molecular weight of the protein components in each fraction. The fractions, which contained compounds of the expected molecular weight, were further analyzed using a capillary zone electrophoresis (CZE) method developed for the routine analysis of wheat gliadins. Using the two CE methods, we were able to compare BM180 with certain gliadin fractions. Additional information on the protein stability was also obtained.

A putative sub-10-kDa basement membrane activity required for lung alveolar formation in vitro.

Basement membrane promotes the reassembly of isolated type II alveolar cells into alveoli-like structures, a process attributable in part to a novel cell adhesion site in the alpha 1-chain of laminin-1 (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The possibility that basement membrane contains other alveolarization activities was probed by subtraction analysis and use of neutralizing antibodies. Deletion of components < 100 kDa, and subsequently < 10 kDa, reduced alveolar cross-sectional area by 70% to 22-25 x 10(3) microns2: the approximate size of alveolar-like structures formed on purified laminin-1 alone. The deleted basement membrane material was adhesive for type II alveolar cells but failed to support alveolar formation in the absence of laminin-1. Preincubation of basement membrane with neutralizing anti-epidermal growth factor (EGF), -basic fibroblast growth factor (bFGF), -insulin-like growth factor (IGF)II, or -transforming growth factor (TGF)-beta antibodies had no inhibitory effect. Because both subtracted basement membrane preparations have in common the exclusion of components < 10 kDa, these results are interpreted as pointing to a sub-10-kDa alveolarization activity(s) that plays a key accessory role in laminin-1-dependent alveolar formation.

"BM180": a novel basement membrane protein with a role in stimulus-secretion coupling by lacrimal acinar cells.

Regulated secretion requires the developmental coupling of neuronal or hormonal stimuli to an exocytotic response, a multistep pathway whose appearance may be linked with cellular adhesion to the newly formed exocrine cell basement membrane. We screened for adhesion-associated coupling activity using lacrimal acinar cells and have identified "BM180", a novel basement membrane protein enriched in guanidine HCl extracts of lacrimal and parotid exocrine secretory glands. BM180 resides primarily in a previously inexamined lower molecular-mass basement membrane peak (peak 2) that contains cell adhesion activity inhibitable with the anti-BM180 monoclonal antibody 3E12. Removal of peak 2 by gel filtration or preincubation of basement membrane with 3E12 decreased regulated peroxidase secretion by one-half without affecting constitutive secretion or the amount of cellular peroxidase available for release. Adding back peak 2 restored regulated secretion in a dose-dependent and 3E12-inhibitable manner and suggested a synergistic relationship between BM180 and laminin 1. BM180 has a mobility of 180 and 60 kDa in the absence or presence of dithiothreitol, respectively, and shows no immunological identity by competitive enzyme-linked immunosorbent assay with laminin 1, collagen IV, entactin, fibronectin, BM-40, perlecan, or vitronectin. We propose that BM180 is an important resident of certain glandular basement membranes where it interacts with the cell surface, thereby possibly signaling the appearance of a transducing element in the stimulus-secretion coupling pathway.

Immunological and partial sequence identity of mouse BM180 with wheat alpha-gliadin.

BM180, a novel 180-kDa basement membrane protein enriched in guanidine-HCl extracts of lacrimal and parotid exocrine secretory glands, was immunopurified using the secretion inhibitory monoclonal antibody 3E12. The N-terminal amino acid sequence was found to be VRVPVPQLQPQNP. An identical sequence comprises the N-terminus of the wheat storage protein alpha-gliadin. The presence of a gliadin-like protein in basement membranes was confirmed using a monoclonal and several polyclonal anti-gliadin antibodies, the former of which detected a 180-kDa protein in basement membrane blots. A full-length alpha-gliadin cDNA was found to hybridize at high stringency with mouse and human genomic DNA; and in lacrimal gland Northern blots with a 2.3-kb message. Since BM180 appears to be required for stimulus-secretion coupling by lacrimal acinar cells, circulating anti-alpha-gliadin antibodies associated with Sjögren's syndrome ('Dry Eye') and more commonly in Coeliac disease, may be secretion inhibitory.

Recombinant domain III of perlecan promotes cell attachment through its RGDS sequence.

Perlecan has been previously been shown to support attachment of a wide variety of cells through interactions of its core protein with the cell surface. The core protein domains involved in cell adhesion are, however, unknown. The laminin-like domain III of murine perlecan contains an RGDS sequence and is a likely candidate for supporting integrin-mediated cell attachment. We made a cDNA construct corresponding to domain III and containing an in frame signal peptide at the 5' end as well as in frame a stop codon at the 3' end by using cDNA clones to perlecan. The construct was inserted into the pRC/CMV vector and transfected into HT1080 cells, and the secreted recombinant domain III, a 130-kDa protein, was purified from the medium. The size of proteolytic fragments produced by digestion with V8 protease as well as analysis of the rotary shadowed image of the recombinant protein indicated it was produced in a native conformation. Recombinant domain III coated on tissue culture dishes, supports adhesion of an epithelial-like mouse mammary tumor cell line MMT 060562 in a dose-dependent manner. This interaction was inhibited specifically by the RGDS synthetic peptide and intact perlecan, but not laminin. This domain III RGD-dependent cell attachment activity indicates a role for perlecan in integrin-mediated signaling.

A novel laminin E8 cell adhesion site required for lung alveolar formation in vitro.

Basement membrane-adherent type II alveolar cells isolated from lung assemble into lumen-containing cellular spheres which retain the correct polarity and thereby approximate the earliest fetal stage of alveolar morphogenesis. The molecular basis of this process, determined in initial experiments to be attributable mainly to the large heterotrimeric glycoprotein laminin, was probed with laminin proteolytic fragments, antibodies, and synthetic peptides. The carboxy-terminal fragment E8, but not equimolar amounts of fragment P1, blocked alveolar formation. To pursue this observation, we used several anti-E8 antibodies and identified one, prepared against A chain residues 2179-2198 ("SN-peptide") from the first loop of the G domain, as inhibitory. These results were confirmed by use of SN-peptide alone and further defined by trypsin digestion of SN-peptide to the sequence SINNNR. This conserved site promoted divalent cation dependent adhesion of both type II alveolar and HT1080 cells, was inhibitable with equimolar amounts of fragment E8 but not P1, and derives from a form of laminin present in fetal alveolar basement membranes. These studies point to an important novel cell adhesion site in the laminin E8 region with a key role in lung alveolar morphogenesis.

Cell-specific expression of LBP-32 mRNA in retina and other locations of newborn mouse eye as revealed by in situ hybridization.

LBP-32 is a cell surface and cytoplasmic protein which is thought to both mediate cell attachment to laminin and play a role in translation initiation. In the present study, antisense RNA for LBP-32 was used to document its cellular mRNA expression pattern in newborn mouse eye. In situ hybridization revealed that LBP-32 was distributed uniformly through the retina as well as over anterior oblique muscle, in corneal and lens epithelial cells and in capillary endothelial cells of the choroid. This unique cell-specific expression raises interesting questions of the role of LBP-32 in eye development.

Elevated 32-kDa LBP and low laminin mRNA expression in developing mouse cerebrum.

Several laminin receptors have been identified, originally a high-affinity 67-kDa laminin binding protein ('LBP-67'), and later galactosyltransferase and the low-affinity but functionally potent integrin receptors. Attempts at obtaining cDNA for LBP-67, although unsuccessful, have given rise to a full-length cDNA coding for an interesting 32-kDa protein, tentatively referred to as '32-kDa LBP', whose relationship to LBP-67 is unclear. Since no information is available on the in vivo expression of 32-kDa LBP mRNA nor of the three laminin chains during CNS development, appropriate 35S-antisense and -sense RNA probes were applied to developing mouse cerebral wall at embryonic day (E)10-16, birth and 1-3 weeks after birth. Expression was examined using Northern blot analysis and in situ hybridization. The 32-kDa LBP mRNA was found to be elevated during the embryonic and perinatal period, and then rapidly declined. At the cellular level, 32-kDa LBP mRNA was distributed throughout the embryonic cerebral wall and became concentrated during the perinatal period in the proliferative ventricular zone and in the cortical plate. By comparison, laminin B1, B2, and A chain mRNA expression was relatively low at all times examined, in keeping with the punctate distribution of laminin antigenicity previously observed by others in developing brain parenchyma. Whereas the functional characterization of 32-kDa LBP and the nature of its laminin and proposed nonlaminin ligands is incomplete, the elevated and unique distribution of 32-kDa LBP mRNA raises interesting questions of the role of 32-kDa LBP mRNA in CNS development.

Altered basement membrane protein biosynthesis by primary cultures of cpk/cpk mouse kidney.

Previously, kidneys from three-week-old cpk/cpk C57/B16 mice were found to contain elevated mRNA levels for the basement membrane components collagen IV and laminin [1]. Here primary cultures of kidney epithelial cells derived from cpk/cpk C57/B16 mice were established and the production of these proteins in culture was studied. Primary cultures of cpk/cpk mouse kidney epithelial cells were observed to have a more polygonal, flattened morphology than cells from unaffected littermate kidneys. The rate of collagen IV and laminin biosynthesis was determined by means of [35S] labelling studies followed by immunoprecipitation. Collagen IV and laminin biosynthesis are elevated by approximately twofold or more in primary cultures derived from 20-day-old cpk/cpk mice, as compared with parallel primary cultures derived from their unaffected littermates. Similarly, laminin B1 chain mRNA is elevated in primary cultures derived from 20-day-old cpk/cpk mice. In primary cultures derived from younger (day 11) mice, similar differences in the rates of both collagen and laminin biosynthesis were not observed between the two culture types. These observations are consistent with the previously reported age-dependent differences observed in laminin and in collagen IV gene expression in both cpk/cpk and wild-type mouse kidneys, and suggest that the regulation of overproduction of these proteins is due to an alteration in the kidney cells and not due to systemic factors.