Did androgen-binding protein paralogs undergo neo- and/or Subfunctionalization as the Abp gene region expanded in the mouse genome?
ABSTRACT: The Androgen-binding protein (Abp) region of the mouse genome contains 30 Abpa genes encoding alpha subunits and 34 Abpbg genes encoding betagamma subunits, their products forming dimers composed of an alpha and a betagamma subunit. We endeavored to determine how many Abp genes are expressed as proteins in tears and saliva, and as transcripts in the exocrine glands producing them. Using standard PCR, we amplified Abp transcripts from cDNA libraries of C57BL/6 mice and found fifteen Abp gene transcripts in the lacrimal gland and five in the submandibular gland. Proteomic analyses identified proteins corresponding to eleven of the lacrimal gland transcripts, all of them different from the three salivary ABPs reported previously. Our qPCR results showed that five of the six transcripts that lacked corresponding proteins are expressed at very low levels compared to those transcripts with proteins. We found 1) no overlap in the repertoires of expressed Abp paralogs in lacrimal gland/tears and salivary glands/saliva; 2) substantial sex-limited expression of lacrimal gland/tear expressed-paralogs in males but no sex-limited expression in females; and 3) that the lacrimal gland/tear expressed-paralogs are found exclusively in ancestral clades 1, 2 and 3 of the five clades described previously while the salivary glands/saliva expressed-paralogs are found only in clade 5. The number of instances of extremely low levels of transcription without corresponding protein production in paralogs specific to tears and saliva suggested the role of subfunctionalization, a derived condition wherein genes that may have been expressed highly in both glands ancestrally were down-regulated subsequent to duplication. Thus, evidence for subfunctionalization can be seen in our data and we argue that the partitioning of paralog expression between lacrimal and salivary glands that we report here occurred as the result of adaptive evolution.
Project description:The house mouse Androgen-binding protein (Abp) gene family is comprised of 64 paralogs, 30 Abpa and 34 Abpbg, encoding the alpha (ABPA) and beta-gamma (ABPBG) protein subunits that are disulfide-bridged to form dimers in secretions. Only 14 Abp genes are expressed in distinct patterns in the lacrimal (11) and submandibular glands (3). We created a knockout mouse line lacking two of the three genes expressed in submandibular glands, Abpa27 and Abpbg27, by replacing them with the neomycin resistance gene. The knockout genotype (-/-) showed no Abpa27 or Abpbg27 transcripts in submandibular gland complementary DNA (cDNA) libraries and there was a concomitant lack of protein expression of ABPA27 and ABPBG27 in the -/- genotype saliva, shown by elimination of these two proteins from the saliva proteome and the loss of cross-reactive material in the acinar cells of the submandibular glands. We also observed a decrease in BG26 protein in the -/- animals, suggesting monomer instability. Overall, we observed no major phenotypic changes in the -/- genotype, compared with their +/+ and +/- siblings raised in a laboratory setting, including normal growth curves, tissue histology, fecundity, and longevity. The only difference is that male and female C57BL/6 mice preferred saliva of the opposite sex containing ABP statistically significantly more than saliva of the opposite sex without ABP in a Y-maze test. These results show for the first time that mice can sense the presence of ABP between saliva targets with and without ABPs, and that they spend more time investigating the target containing ABP.
Project description:Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltration of salivary and lacrimal glands resulting in diminished production of saliva and tears. The pathophysiology of SS has not yet been fully deciphered. Classically it has been postulated that sicca symptoms in SS patients are a double step process whereby lymphocytic infiltration of lacrimal and salivary glands (SG) is followed by epithelial cell destruction resulting in keratoconjunctivitis sicca and xerostomia. Recent advances in the field of the pathophysiology of SS have brought in new players, such as aquaporins (AQPs) and anti AQPs autoantibodies that could explain underlying mechanistic processes and unveil new pathophysiological pathways offering a deeper understanding of the disease. In this review, we delineate the link between the AQP and SS, focusing on salivary glands, and discuss the role of AQPs in the treatment of SS-induced xerostomia.
Project description:Sjögren syndrome is an autoimmune disease characterized by targeted destruction of the lacrimal and salivary glands resulting in symptoms of severe ocular and oral dryness. Despite its prevalence, the mechanisms driving autoimmune manifestations are unclear. In patients and in the nonobese diabetic (NOD) mouse model of Sjögren syndrome, lymphocytic infiltrates consist of CD4 and CD8 T cells, although the role of CD8 T cells in disease pathogenesis has been largely unexplored. Here, we evaluated the contribution of CD8 T cells to lacrimal and salivary gland autoimmunity. Within the lacrimal and salivary glands of NOD mice, CD8 T cells were proliferating, expressed an activated phenotype, and produced inflammatory cytokines. Transfer of purified CD8 T cells isolated from the cervical lymph nodes (LNs) of NOD mice into NOD-severe combined immunodeficiency recipients resulted in inflammation of the lacrimal glands, but was not sufficient to cause inflammation of the salivary glands. Lacrimal gland-infiltrating CD8 T cells displayed a cytotoxic phenotype, and epithelial cell damage in the lacrimal glands was observed in recipients of CD8 T cells regardless of the presence of CD4 T cells. Collectively, our results demonstrate that CD8 T cells have a pathogenic role in lacrimal gland autoimmunity. The gland-specific pathogenicity of CD8 T cells makes them a valuable resource to further understand the mechanisms that discriminate lacrimal versus salivary gland autoimmunity and for the development of new therapeutics that target the early stages of disease.
Project description:The tear film is a complex mixture of secreted fluid, ions, proteins, glycoproteins, and lipids that lubricates and protects the ocular surface. Recently, several antimicrobial peptides have been described in the tear fluid. In this study, we describe the presence of the large secreted glycoprotein gp340 in the tear film. Western blot analysis showed that gp340 is abundant in secreted tears and in the lacrimal glands. Lesser amounts of gp340 were detected in the cornea and conjunctiva. Consistent with Western blot data, reverse transcription-PCR and real-time quantitative PCR showed that gp340 transcripts were abundant in lacrimal gland tissue and were also present in the cornea and conjunctiva. Immunohistochemistry localized gp340 to the acinar cells of the lacrimal gland and the deeper layers of the conjunctival epithelium. gp340 was not detected in conjunctival goblet cells. In the cornea, gp340 was present only in a peripheral band of basal epithelial cells, suggesting that gp340 may play a role in the cycle of corneal epithelial renewal. To determine if tear film gp340 may function as a bacterial agglutinin as it does in saliva, tears were incubated with streptococcal cells and the formation of bacterial aggregates was monitored. Addition of tears to late-exponential-phase Streptococcus mutans cells resulted in time- and dose-dependent aggregation of the bacteria. Furthermore, Western blot analysis confirmed the presence of cell-associated gp340 in isolated bacterial aggregates. The ocular pathogen Staphylococcus aureus, but not Pseudomonas aeruginosa, also aggregated when incubated with tears. These results suggest that gp340 is a normal component of the tear film and that the glycoprotein may function as a bacterial agglutinin.
Project description:Generalist herbivores feed on a wide array of plants and need to adapt to varying host qualities and defenses. One of the first insect derived secretions to come in contact with the plant is the saliva. Insect saliva is potentially involved in both the pre-digestion of the host plant as well as induction/suppression of plant defenses, yet how the salivary glands respond to changes in host plant at the transcriptional level is largely unknown. The objective of this study was to determine how the labial salivary gland transcriptome varies according to the host plant on which the insect is feeding. In order to determine this, cabbage looper (Trichoplusia ni) larvae were reared on cabbage, tomato, and pinto bean artificial diet. Labial glands were dissected from fifth instar larvae and used to extract RNA for RNASeq analysis. Assembly of the resulting sequencing reads resulted in a transcriptome library for T. ni salivary glands consisting of 14,037 expressed genes. Feeding on different host plant diets resulted in substantial remodeling of the gland transcriptomes, with 4,501 transcripts significantly differentially expressed across the three treatment groups. Gene expression profiles were most similar between cabbage and artificial diet, which corresponded to the two diets on which larvae perform best. Expression of several transcripts involved in detoxification processes were differentially expressed, and transcripts involved in the spliceosome pathway were significantly downregulated in tomato-reared larvae. Overall, this study demonstrates that the transcriptomes of the salivary glands of the cabbage looper are strongly responsive to diet. It also provides a foundation for future functional studies that can help us understand the role of saliva of chewing insects in plant-herbivore interactions.
Project description:Nonobese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity similar to human Sjögren syndrome. In both humans and NOD mice, the early immune response that drives T-cell infiltration into lacrimal and salivary glands is poorly understood. In NOD mice, lacrimal gland autoimmunity spontaneously occurs only in males with testosterone playing a role in promoting lacrimal gland inflammation, while female lacrimal glands are protected by regulatory T cells (Tregs). The mechanisms of this male-specific lacrimal gland autoimmunity are not known. Here, we studied the effects of Treg depletion in hormone-manipulated NOD mice and lacrimal gland gene expression to determine early signals required for lacrimal gland inflammation. While Treg-depletion was not sufficient to drive dacryoadenitis in castrated male NOD mice, chemokines (Cxcl9, Ccl19) and other potentially disease-relevant genes (Epsti1, Ubd) were upregulated in male lacrimal glands. Expression of Cxcl9 and Ccl19, in particular, remained significantly upregulated in the lacrimal glands of lymphocyte-deficient NOD-severe combined immunodeficiency (SCID) mice and their expression was modulated by type I interferon signaling. Notably, Ifnar1-deficient NOD mice did not develop dacryoadenitis. Together these data identify disease-relevant genes upregulated in the context of male-specific dacryoadenitis and demonstrate a requisite role for type I interferon signaling in lacrimal gland autoimmunity in NOD mice.
Project description:Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in lacrimal gland epithelium cells and demonstrated that the simultaneous overexpression of these transcription factors can differentiate human embryonic stem cells into lacrimal gland epithelium-like cells. This study suggests the possibility of lacrimal glands regeneration from human pluripotent stem cells.
Project description:Inflammation and impaired secretion by lacrimal and salivary glands are hallmarks of the autoimmune disease, Sjögren's Syndrome. These changes in the lacrimal gland promote dryness and inflammation of the ocular surface, causing pain, irritation and corneal damage. The changes that initiate and sustain autoimmune inflammation in the lacrimal gland are not well-established. Here we demonstrate that interferon-? (IFN-?) is significantly elevated in lacrimal gland and tears of the male NOD mouse, a model of autoimmune dacryoadenitis which exhibits many ocular characteristics of Sjögren's Syndrome, by 12 weeks of age early in lacrimal gland inflammation. Working either with primary cultured lacrimal gland acinar cells from BALB/c mice and/or rabbits, in vitro IFN-? treatment for 48 hr decreased expression of Rab3D concurrent with increased expression of cathepsin S. Although total cellular cathepsin S activity was not commensurately increased, IFN-? treated lacrimal gland acinar cells showed a significant increase in carbachol-stimulated secretion of cathepsin S similar to the lacrimal gland in disease. In vitro IFN-? treatment did not increase the expression of most components of major histocompatibility complex (MHC) class II-mediated antigen presentation although antigen presentation was slightly but significantly stimulated in primary cultured lacrimal gland acinar cells. However, exposure of cultured human corneal epithelial cells to IFN-? more robustly increased expression and activity of cathepsin S in parallel with increased expression and function of MHC class II-mediated antigen presentation. We propose that early elevations in IFN-? contribute to specific features of ocular disease pathology in Sjögren's Syndrome.
Project description:The lacrimal gland (LG) develops through branching morphogenesis and produces secretions, including tears, that lubricate and protect the ocular surface. Despite the prevalence of LG disorders such as dry eye, relatively little is known about the regulation of LG development. In this study, we show that the homeobox transcription factor Barx2 is highly expressed in conjunctival epithelium, eyelids and ocular [lacrimal, harderian (HG), and meibomian (MG)] glands and is necessary for normal ocular gland and eyelid development. Barx2(-/-) mice show defective LG morphogenesis, absence of the HG, and defects in MG and eyelid fusion. Ex vivo antisense assays confirm the requirement for Barx2 in LG bud elongation and branching. Gene expression profiles reveal decreased expression of several adhesion and matrix remodeling molecules in Barx2(-/-) LGs. In culture, Barx2 regulates expression of matrix metalloproteinases (MMPs) and epithelial cell migration through the extracellular matrix. Fibroblast growth factors are crucial regulators of LG development and we show that Barx2 is required for Fgf10-induced LG bud elongation and that both Barx2 and Fgf10 cooperate in the regulation of MMPs. Together, these data suggest a mechanism for the effects of loss of Barx2 on ocular gland development. Intriguingly, salivary glands that also express a high level of Barx2 develop normally in Barx2(-/-) mice and do not show altered levels of MMPs. Thus, the function of Barx2 is specific to ocular gland development. Based on our data, we propose a functional network involving Barx2, Fgf10 and MMPs that plays an essential role in regulating branching morphogenesis of the ocular glands.
Project description:Aqueous tear-deficient dry eye is a multifactorial chronic disorder in which the lacrimal glands fail to produce enough tears to maintain a healthy ocular surface. The existence of lacrimal gland stem/progenitor cells was proposed in several species, yet their origin and characteristics are not very clear. Here, we investigated the presence of resident progenitor cells and their regenerative potential in a rabbit model with lacrimal gland main excretory duct ligation-induced injury. The ligation-injured lacrimal glands temporarily decreased in weight and had impaired tear secretion. Protein expression profiles and transcriptional profiles were obtained from injured tissue. Isolated lacrimal gland progenitor cells were tested and characterized by stem cell-related marker evaluation, single cell clonal assay and three-dimensional (3-D) culture. The results of our study indicate that lacrimal glands are capable of tissue repair after duct ligation-induced injury, likely involving resident stem/progenitor cells and epithelial-mesenchymal transitions. Lacrimal gland progenitor cells isolated from ligated tissue can differentiate in 3-D culture. The results provide further insights into lacrimal gland stem/progenitor cell physiology and their potential for treating severe cases of tear deficiency.