Enteric viruses replicate in salivary glands and infect through saliva.

Enteric viruses like norovirus, rotavirus and astrovirus have long been accepted as spreading in the population through fecal-oral transmission: viruses are shed into feces from one host and enter the oral cavity of another, bypassing salivary glands (SGs) and reaching the intestines to replicate, be shed in feces and repeat the transmission cycle1. Yet there are viruses (for example, rabies) that infect the SGs2,3, making the oral cavity one site of replication and saliva one conduit of transmission. Here we report that enteric viruses productively and persistently infect SGs, reaching titres comparable to those in the intestines. We demonstrate that enteric viruses get released into the saliva, identifying a second route of viral transmission. This is particularly significant for infected infants, whose saliva directly transmits enteric viruses to their mothers' mammary glands through backflow during suckling. This sidesteps the conventional gut-mammary axis route4 and leads to a rapid surge in maternal milk secretory IgA antibodies5,6. Lastly, we show that SG-derived spheroids7 and cell lines8 can replicate and propagate enteric viruses, generating a scalable and manageable system of production. Collectively, our research uncovers a new transmission route for enteric viruses with implications for therapeutics, diagnostics and importantly sanitation measures to prevent spread through saliva.

Epithelial Cell Lineage and Signaling in Murine Salivary Glands.

Maintaining salivary gland function is critical for oral health. Loss of saliva is a common side effect of therapeutic irradiation for head and neck cancer or autoimmune diseases such as Sjögren's syndrome. There is no curative treatment, and current strategies proposed for functional regeneration include gene therapy to reengineer surviving salivary gland tissue, cell-based transplant therapy, use of bioengineered glands, and development of drugs/biologics to stimulate in vivo regeneration or increase secretion. Understanding the genetic and cellular mechanisms required for development and homeostasis of adult glands is essential to the success of these proposed treatments. Recent advances in genetic lineage tracing provide insight into epithelial lineage relationships during murine salivary gland development. During early fetal gland development, epithelial cells expressing keratin 14 (K14) Sox2, Sox9, Sox10, and Trp63 give rise to all adult epithelium, but as development proceeds, lineage restriction occurs, resulting in separate lineages of myoepithelial, ductal, and acinar cells in postnatal glands. Several niche signals have been identified that regulate epithelial development and lineage restriction. Fibroblast growth factor signaling is essential for gland development, and other important factors that influence epithelial patterning and maturation include the Wnt, Hedgehog, retinoic acid, and Hippo signaling pathways. In addition, other cell types in the local microenvironment, such as endothelial and neuronal cells, can influence epithelial development. Emerging evidence also suggests that specific epithelial cells will respond to different types of salivary gland damage, depending on the cause and severity of damage and the resulting damaged microenvironment. Understanding how regeneration occurs and which cell types are affected, as well as which signaling factors drive cell lineage decisions, provides specific targets to manipulate cell fate and improve regeneration. Taken together, these recent advances in understanding cell lineages and the signaling factors that drive cell fate changes provide a guide to develop novel regenerative treatments.

Neurturin-containing laminin matrices support innervated branching epithelium from adult epithelial salispheres.

Cell transplantation of autologous adult biopsies, grown ex vivo as epithelial organoids or expanded as spheroids, are proposed treatments to regenerate damaged branching organs. However, it is not clear whether transplantation of adult organoids or spheroids alone is sufficient to initiate a fetal-like program of branching morphogenesis in which coordinated branching of multiple cell types including nerves, mesenchyme and blood vessels occurs. Yet this is an essential concept for the regeneration of branching organs such as lung, pancreas, and lacrimal and salivary glands. Here, we used factors identified from fetal organogenesis to maintain and expand adult murine and human epithelial salivary gland progenitors in non-adherent spheroid cultures, called salispheres. These factors stimulated critical developmental pathways, and increased expression of epithelial progenitor markers such as Keratin5, Keratin14, FGFR2b and KIT. Moreover, physical recombination of adult salispheres in a laminin-111 extracellular matrix with fetal salivary mesenchyme, containing endothelial and neuronal cells, only induced branching morphogenesis when neurturin, a neurotrophic factor, was added to the matrix. Neurturin was essential to improve neuronal survival, axon outgrowth, innervation of the salispheres, and resulted in the formation of branching structures with a proximal-distal axis that mimicked fetal branching morphogenesis, thus recapitulating organogenesis. Epithelial progenitors were also maintained, and developmental differentiation programs were initiated, showing that the fetal microenvironment provides a template for adult epithelial progenitors to initiate branching and differentiation. Further delineation of secreted and physical cues from the fetal niche will be useful to develop novel regenerative therapies that instruct adult salispheres to resume a developmental-like program in vitro and to regenerate branching organs in vivo.

Viral gene transfer to developing mouse salivary glands.

Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.

Salivary gland progenitor cell biology provides a rationale for therapeutic salivary gland regeneration.

An irreversible loss of salivary gland function often occurs in humans after removal of salivary tumors, after therapeutic radiation of head and neck tumors, as a result of Sjögren's syndrome and in genetic syndromes affecting gland development. The permanent loss of gland function impairs the oral health of these patients and broadly affects their quality of life. The regeneration of functional salivary gland tissue is thus an important therapeutic goal for the field of regenerative medicine and will likely involve stem/progenitor cell biology and/or tissue engineering approaches. Recent reports demonstrate how both innervation of the salivary gland epithelium and certain growth factors influence progenitor cell growth during mouse salivary gland development. These advances in our understanding suggest that developmental mechanisms of mouse salivary gland development may provide a paradigm for postnatal regeneration of both mice and human salivary glands. Herein, we will discuss the developmental mechanisms that influence progenitor cell biology and the implications for salivary gland regeneration.

Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis.

The maintenance of a progenitor cell population as a reservoir of undifferentiated cells is required for organ development and regeneration. However, the mechanisms by which epithelial progenitor cells are maintained during organogenesis are poorly understood. We report that removal of the parasympathetic ganglion in mouse explant organ culture decreased the number and morphogenesis of keratin 5-positive epithelial progenitor cells. These effects were rescued with an acetylcholine analog. We demonstrate that acetylcholine signaling, via the muscarinic M1 receptor and epidermal growth factor receptor, increased epithelial morphogenesis and proliferation of the keratin 5-positive progenitor cells. Parasympathetic innervation maintained the epithelial progenitor cell population in an undifferentiated state, which was required for organogenesis. This mechanism for epithelial progenitor cell maintenance may be targeted for organ repair or regeneration.

Environmental factors affecting feed intake of steers in different housing systems in the summer.

A total of 188 yearling steers of predominantly Angus and Hereford breeds, with mean body weight of 299 kg, were used in this study, which started on 8 April and finished on 3 October, to assess the effects of environmental factors on feed intake of steers in various housing systems. Housing consisted of outside lots with access to overhead shelter, outside lots with no overhead shelter and a cold confinement building. Ad libitum corn, 2.27 kg of 35% dry matter whole plant sorghum silage and 0.68 kg of a 61% protein-vitamin-mineral supplement was offered. Feed that was not consumed was measured to determine feed intake. The temperature data were recorded by hygro-thermographs. Hourly temperatures and humidity were used to develop weather variables. Regression analysis was used and weather variables were regressed on dry matter intake (DMI). When addition of a new variable did not improve R (2) more than one unit, then the number of variables in the model was truncated. Cattle in confinement had lower DMI than those in open lots and those in open lots with access to an overhead shelter (P < 0.05). Cattle in outside lots with access to overhead shelter had similar DMI compared to those in open lots (P = 0.065). Effect of heat was predominantly displayed in August in the three housing systems. In terms of explaining variation in DMI, in outside lots with access to overhead shelter, average and daytime temperatures were important factors, whereas in open lots, nocturnal, peak and average temperatures were important factors. In confinement buildings, the previous day's temperature and humidity index were the most important factors explaining variation in DMI. Results show the effect of housing and weather variables on DMI in summer and when considering these results, cattle producers wishing to improve cattle feedlot performance should consider housing conditions providing less stress or more comfort.

Analysis of cytoskeletal and motility proteins in the sea urchin genome assembly.

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.

Association between increased CCL2 (MCP-1) expression in lesions and persistence of disease activity in giant-cell arteritis.

OBJECTIVE: Patients with giant-cell arteritis (GCA) usually respond dramatically to corticosteroid treatment. However, recurrences are frequent and corticosteroid requirements are highly variable among patients. The aim of our study was to identify genes potentially involved in disease persistence. METHODS: Gene expression was explored with cDNA arrays in temporal artery biopsies from six GCA patients with relapsing disease and six patients who easily achieved sustained remission. Differentially expressed genes of interest were subsequently analysed by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry in temporal artery biopsies from 35 patients with biopsy-proven GCA and nine controls. RESULTS: CCL2 (MCP-1) was up-regulated in temporal artery samples from relapsing individuals. In the extended series of patients, CCL2 mRNA concentration in lesions was significantly higher than in controls (31 +/- 15.6 vs 0.44 +/- 0.10, P = 0.0001). In addition, CCL2 was more abundant in patients who experienced two or more relapses during the first year compared with those who endured sustained remission (127 +/- 82 vs 11 +/- 5.5, P = 0.0233) and correlated with the cumulated prednisolone dose (R = 0.533, P = 0.0024). CCL2 mRNA concentration correlated with IL-1beta (R = 0.45, P = 0.02), tumour necrosis factor-alpha (TNF-alpha) (R = 0.47, P = 0.013) and IL-6 (R = 0.52, P = 0.0053) mRNA. However, circulating CCL2 determined by ELISA was decreased in patients with strong systemic inflammatory response, suggesting that reduction in circulating CCL2 may reinforce the local gradient in lesions. CONCLUSION: Increased CCL2 (MCP-1) expression in lesions is associated with persistence of disease activity in GCA.

Laminin-1 and SIKVAV a laminin-1-derived peptide, regulate the morphology and protease activity of a human salivary gland adenoid cystic carcinoma cell line.

In a previous paper, we demonstrated that laminin-1 and its derived peptide SIKVAV modulates the morphology of an adenoid cystic carcinoma cell line (CAC2 cells). Light microscopy of CAC2 cells grown in three-dimensional preparations of SIKVAV-enriched laminin-1 showed the presence of pseudocystic spaces. Pseudocysts are hallmarks of adenoid cystic carcinoma in vivo. We hypothesized that these pseudocystic spaces could be due to the protease-inducing/activating role of SIKVAV. Thus, we studied the presence of matrix metalloproteinases (MMPs) in CAC2 cells treated either by laminin-1 or by SIKVAV-enriched laminin-1. Immunohistochemistry and zymography suggested that SIKVAV enhanced the secretion of MMP-2 and MMP-9 in CAC2 cells. We propose that SIKVAV induces pseudocystic formation probably through the secretion of MMPs 2 and 9.

NOVH increases MMP3 expression and cell migration in glioblastoma cells via a PDGFR-alpha-dependent mechanism.

Nephroblastoma overexpressed gene (NOV) is highly expressed in the nervous system. We investigated its biological activity by expressing the human NOV gene (NOVH) in a human glioblastoma cell line that is negative for NOVH and by analyzing four clones with different levels of NOVH expression. There was no difference in cell proliferation between the NOVH-expressing cell lines, but there was increased cell adhesion and migration that correlated with increasing NOVH expression. Gene expression profiling was used to investigate the mechanisms by which NOVH expression regulated cell activity. We identified two induced genes in NOVH-expressing cells that are involved in cell migration: matrix metalloprotease (MMP)3 and platelet-derived growth factor receptor (PDGFR)-alpha. Our studies show that PDGFR-alpha induced MMP3 gene expression and increased cell proliferation and cell migration upon stimulation by platelet-derived growth factor (PDGF)-AA. We also show that the induction of MMP3 in cells expressing NOVH is potentiated by either cell density, serum, or PDGF-BB. Thus, expression of NOVH in glioblastoma cells triggers a cascade of gene expression resulting in increased cell adhesion and migration.

Cell type-specific differences in glycosaminoglycans modulate the biological activity of a heparin-binding peptide (RKRLQVQLSIRT) from the G domain of the laminin alpha1 chain.

AG73 (RKRLQVQLSIRT), a peptide from the G domain of the laminin alpha1 chain, has diverse biological activities with different cell types. The heparan sulfate side chains of syndecan-1 on human salivary gland cells were previously identified as the cell surface ligand for AG73. We used homologous peptides from the other laminin alpha-chains (A2G73-A5G73) to determine whether the bioactivity of the AG73 sequence is conserved. Human salivary gland cells and a mouse melanoma cell line (B16F10) both bind to the peptides, but cell attachment was inhibited by glycosaminoglycans, modified heparin, and sized heparin fragments in a cell type-specific manner. In other assays, AG73, but not the homologous peptides, inhibited branching morphogenesis of salivary glands and B16F10 network formation on Matrigel. We identified residues critical for AG73 bioactivity using peptides with amino acid substitutions and truncations. Fewer residues were critical for inhibiting branching morphogenesis (XKXLXVXXXIRT) than those required to inhibit B16F10 network formation on Matrigel (N-terminal XXRLQVQLSIRT). In addition, surface plasmon resonance analysis identified the C-terminal IRT of the sequence to be important for heparin binding. Structure-based sequence alignment predicts AG73 in a beta-sheet with the N-terminal K (Lys(2)) and the C-terminal R (Arg(10)) on the surface of the G domain. In conclusion, we have determined that differences in cell surface glycosaminoglycans and differences in the amino acids in AG73 recognized by cells modulate the biological activity of the peptide and provide a mechanism to explain its cell-specific activities.

PKC and ERK1/2 regulate amylase promoter activity during differentiation of a salivary gland cell line.

The addition of transforming growth factor alpha (TGFalpha) to a human submandibular gland cell line (HSG) cultured on basement membrane extract Matrigel, synergistically activates the acinar cell-specific salivary amylase promoter. Signaling through beta1 integrins and increased phosphorylation of ERK1/2 are involved in the increased promoter activity. Phorbol-12-myristate-13-acetate (PMA) and thapsigargin increase amylase promoter activity, suggesting that phorbol ester and calcium-dependent protein kinase C (PKC) pathways are also involved. The combination of specific inhibitors of PKC and MEK1 inhibits the amylase promoter. Inhibitors of the calcium-dependent PKC isoforms alpha, beta, and gamma decrease the promoter activity; however, PKCbeta is not detectable in HSG cells. TGFalpha alters the cellular localization of PKCalpha but not -gamma, suggesting PKCalpha is involved in TGFalpha upregulation of the amylase promoter. Furthermore, rottlerin, a PKCdelta-specific inhibitor, increases the promoter activity, suggesting PKC isoforms differentially regulate the amylase promoter. In conclusion, beta1-integrin and TGFalpha signaling pathways regulate the amylase promoter activity in HSG cells. In response to Matrigel and TGFalpha, the activation of both PKCalpha and phosphorylation of ERK1/2 results in synergistic activation of the amylase promoter. Published 2000 Wiley-Liss, Inc.

Cell adhesive sequences in mouse laminin beta1 chain.

Laminin-1, a major component of the basement membrane, consists of three different chains, alpha1, beta1, and gamma1. We sought to identify cell adhesive sequences from the mouse laminin beta1 chain by testing HT-1080 fibrosarcoma and B16-F10 melanoma cells for binding to 187 overlapping synthetic peptides which covered the entire chain. Fourteen peptides showed cell adhesive activities with either peptide-conjugated Sepharose beads or peptide-coated plates or both. Additional cells, including neuronal, endothelial, and salivary gland cells, showed biological responses in a cell type-specific manner. B-7, B-133, and B-160 showed the most potent cell attachment. Cell binding on three peptides (B-34, B-133, and B-160) was inhibited by EDTA. Cell adhesion to 11 of the 12 active peptides was inhibited to varying degrees by heparin. Of the 17 active peptides identified in the laminin beta1 chain in this and other studies, 8 are clustered on the amino terminal globular domain, suggesting a possible important role in cell binding for this domain that may be multifunctional. These data demonstrate that the laminin beta1 chain has multiple active sites for cell adhesion, some of which are cell-type specific.

Identification of a major heparin and cell binding site in the LG4 module of the laminin alpha 5 chain.

The G domain of the laminin alpha chains consists of five homologous G modules (LG1-5) and has been implicated in various biological functions. In this study, we identified an active site for cell and heparin binding within the laminin alpha5 G domain using recombinant proteins and synthetic peptides. Recombinant LG4, LG5, and LG4-5 modules were generated using a mammalian expression system. The LG4 and LG4-5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inhibited cell binding to the LG4-5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin beta(1) subunit. These results suggest that the LG4-5 module interacts with a cell surface receptor containing heparan sulfate but not with integrins. Solid-phase assays and surface plasmon resonance measurements demonstrated strong binding of the LG4 and LG4-5 modules to heparin with K(D) values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with glycosidases demonstrated that N-linked carbohydrates on the LG5 module are complex-type oligosaccharides. The LG4-5 module, devoid of N-linked carbohydrates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal of N-linked glycosylation. To localize active sites on the LG4 module, various synthetic peptides were used to compete with binding of the tandem module to heparin and cells. Peptide F4 (AGQWHRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to compete binding. Alanine replacements demonstrated that one arginine residue within F4 was important for cell and heparin binding. Our results suggest a critical role of the LG4 module for heparan sulfate-containing receptor binding within the laminin alpha5 chain.

Identification of endothelial cell binding sites on the laminin gamma 1 chain.

The laminins belong to a family of trimeric basement membrane glycoproteins with multiple domains, structures, and functions. Endothelial cells bind laminin-1 and form capillary-like structures when plated on a laminin-1-rich basement membrane matrix, Matrigel. Laminin-1 is composed of 3 chains, alpha1, beta1, and gamma1. Because laminin-1 is known to contain multiple biologically active sites, we have screened 156 synthetic overlapping peptides spanning the entire laminin gamma1 chain for potential angiogenic sequences. Only 7 of these peptides, designated as C16, C25, C30, C38, C64, C75, and C102, disrupted the formation of capillary-like structures by human umbilical vein endothelial cells on Matrigel. Dose-response experiments in the presence of 50 to 200 microg/mL showed that tube formation was prevented by most peptides at 150 and 200 microg/mL, except for C16, which showed strong activity at all concentrations. Active peptides promoted vessel sprouting from aorta rings and angiogenesis in the chick chorioallantoic membrane assay. In addition, the active peptides also promoted endothelial cell adhesion to dishes coated with 0.1 microg of peptide and inhibited attachment to laminin-1 but not to plastic or fibronectin. Four of the active peptides, C25, C38, C75, and C102, may have cell-type specificity with endothelial cells, since they did not promote PC12 neurite outgrowth or adhesion of B16-F10 melanoma and human submandibular gland cells. These results suggest that specific laminin gamma1-chain peptides have angiogenic activity with potential therapeutic applications.

Cell binding sequences in mouse laminin alpha1 chain.

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.

Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line.

The culture of human submandibular gland (HSG) cells on laminin-1 induces acinar differentiation. We identified a site on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin alpha1 and alpha2 chains. The alpha1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on laminin-1. Cells cultured with either AG73 or the homologous alpha2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on laminin are required for complete differentiation. The G-domain of laminin-1 contains both integrin and heparin binding sites, and anti-beta1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of laminin-1 containing AG73, is specific, since other laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-beta1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of laminin-1 and laminin-2. In summary, multiple interactions between laminin and HSG cells contribute to acinar differentiation, involving both beta1-integrins and syndecan-1.

Growth factor regulation of the amylase promoter in a differentiating salivary acinar cell line.

Salivary glands contain two major epithelial cell types: acinar cells which produce the primary salivary secretion, including amylase, and ductal cells which reabsorb electrolytes but also secrete kallikrein. Here we investigated salivary acinar cell differentiation in vitro using the activity of the salivary amylase and tissue kallikrein promoters as markers of acinar cell and ductal cell differentiation, respectively. Each of the promoter sequences was cloned into a replication-deficient adenoviral vector containing the luciferase reporter gene. Previous studies showed that a human submandibular gland cell line (HSG) differentiated into acinar cells when cultured on a reconstituted basement membrane matrix (Matrigel). The luciferase activity of the amylase promoter vector (AdAMY-luc) was low in HSG cells cultured on plastic, where they grow as an epithelial monolayer. The promoter activity increased approximately tenfold when HSG cells were cultured on Matrigel and developed an acinar phenotype. Under the same conditions, the luciferase activity of the kallikrein promoter (AdKALL-luc) was not induced. Because HSG cells demonstrate acinar cell morphology, but not amylase gene expression, when cultured on laminin-1, certain soluble components of Matrigel were tested for their ability to induce the amylase promoter during in vitro differentiation of acinar cells. We find that epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), which are present in the basement membrane, and hepatocyte growth factor (HGF) increase activity of the amylase promoter. Other basement membrane-derived growth factors such as TGF-beta, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PGDF), as well as tumor necrosis factor (TNF-alpha), keratinocyte growth factor (KGH), nerve growth factor (NGF) and interferon gamma (IFN-gamma) were inactive. This system will be further exploited to study the mechanisms by which extracellular matrix molecules and growth factors regulate salivary acinar cell differentiation.

Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells.

Invasive prostatic carcinomas and prostatic intraepithelial neoplasia (PIN) are characterized by a loss of normal cell organization, cell polarity, and cell:cell and cell:basement membrane adhesion. The objective of this study was to establish in vitro three-dimensional (3-D) cell models which can be used to investigate mechanisms involved in acinar morphogenesis and differentiation in normal prostatic epithelium and their abnormalities in cancer cells. The process of acinar morphogenesis, including structural and functional differentiation, was investigated by culture on basement membrane gels (Matrigel). The human papillomavirus 18 immortalized, non-tumorigenic cell line RWPE-1, the v-Ki-ras transformed, tumorigenic RWPE-2 cell line derived from RWPE-1 cells (see previous paper pp. 1221-1229) and the human prostatic carcinoma cell line DU-145 were used. When cultured on Matrigel, RWPE-2 cells remain as single cells or form small aggregates and DU-145 cells form large amorphous cell aggregates without any organization or lumen. In contrast, RWPE-1 cells form acini of polarized epithelium with a distinct lumen, show a distinct laminin basement membrane, and express alpha6beta1 integrins at their basal end. Exposure to conditioned medium from NIH 3T3 cultures accelerates glandular morphogenesis. Parallel cultures maintained as monolayers on plastic remain as monolayers. In the presence of the synthetic androgen mibolerone, acinar cells express prostate specific antigen (PSA) as determined by immunostaining. We conclude that normal prostate cells can undergo acinar morphogenesis while tumorigenic cells have lost this ability. The 3-D cultures provide physiologically relevant in vitro models for elucidating regulation of growth, morphogenesis and differentiation in the normal human prostate, for defining heterotypic interactions in benign prostatic hyperplasia and for establishing the basis for the loss of normal cell organization in early neoplastic lesions such as PIN as well as during tumor progression in prostate cancer.