Mouse Lacrimal Gland Research Articles

Activation of HIF-1α (hypoxia inducible factor-1α) prevents dry eye-induced acinar cell death in the lacrimal gland.

The pathogenesis of immune-mediated lacrimal gland (LG) dysfunction in Sjögren's syndrome has been thoroughly studied. However, the majority of dry eye (DE) is not related to Sjögren type, and its pathophysiology remains unclear. The purpose of this study was to determine and investigate the protective mechanisms against DE stress in mice. DE induced prominent blood vessel loss without apoptosis or necrosis in the LG. Autophagic vacuoles, distressed mitochondria, and stressed endoplasmic reticulum were observed via electron microscopy. Immunoblotting confirmed the increase in autophagic markers. Glycolytic activities were enhanced with increasing levels of succinate and malate that, in turn, activated hypoxia-inducible factor (HIF)-1α. Interestingly, the areas of stable HIF-1α expression overlapped with COX-2 and MMP-9 upregulation in LGs of DE-induced mice. We generated HIF-1α conditional knockout (CKO) mice in which HIF-1α expression was lost in the LG. Surprisingly, normal LG polarities and morphologies were completely lost with DE induction, and tremendous acinar cell apoptosis was observed. Similar to Sjögren's syndrome, CD3+ and CD11b+ cells infiltrated HIF-1α CKO LGs. Our results show that DE induced the expression of HIF-1α that activated autophagy signals to prevent further acinar cell damage and to maintain normal LG function.

Necessity of Smad4 for the normal development of the mouse lacrimal gland

Smad4, a key intracellular mediator in TGF-β signaling, plays a critical role in the normal development of many tissues/organs. However, its functional role in the development of the lacrimal gland (LG) has never been reported. The aim of this study was to investigate the role Smad4 may play in the development of LG by using Smad4 conditional knockout mice and comparing their LG changes with the LG in normal control mice. Smad4 in the LG, as well as in the lens, cornea, and ectoderm of the eyelids, was conditionally inactivated by using Pax6 promoter-driven Cre-transgenic mice. Lac Z reporter was used to visualize the developing LG by X-gal staining, and standard histologic approaches were used to reveal morphologic alterations. Inactivation of Smad4 resulted in reduction in the size and number of acini in the embryonic LG and in pigment accumulation within the LG, although it did not affect the formation of the primary lacrimal bud. After birth, the LG from Smad4-mutant mice developed slowly, and pigment was observed starting from the P7 mutant LG. Thereafter, the mutant LG was considerably smaller than the normal LG and was eventually replaced by adipose tissue. These results support the notion that Smad4 is essential for the normal development and maintenance of the mouse LG.

Role of Orai1 and store‐operated calcium entry in mouse lacrimal gland signalling and function

Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function.

Presence and Physiologic Function of the Renin–Angiotensin System in Mouse Lacrimal Gland

To investigate the expression, localization, and physiologic function of renin-angiotensin system (RAS) components in the mouse lacrimal gland. Lacrimal glands and cultured lacrimal gland fibroblasts from wild-type (WT) BALB/c (H-2(d)) mice were used. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to determine the expression and localization of the RAS components, prorenin/renin, angiotensin-converting enzyme (ACE), angiotensin II, angiotensin II type 1 receptor (AT1R), and angiotensin II type 2 receptor (AT2R) in the normal mouse lacrimal gland. To examine the change in tear secretion, mice received ARB (AT1R blocker) or AT2R antagonist. Tear secretion was assessed by cotton thread test before and after drug administration. The mRNAs coding for angiotensinogen, prorenin, ACE, and both AT1R and AT2R were found in normal lacrimal gland tissue and cultured lacrimal gland fibroblasts. Prorenin/renin and ACE were identified in myoepithelial cells around ducts and acini and in blood vessels. Angiotensin II, AT1R, and AT2R were observed in the ducts and interstitial fibroblasts. AT1R and AT2R were also localized in blood vessels. All the cultured lacrimal gland fibroblasts expressed angiotensin II, AT1R, and AT2R. Tear secretion increased in mice that received ARB. The results are consistent with the hypothesis that a tissue-specific RAS is present in the lacrimal gland, and suggest that fibroblasts are one of the cell types playing a role in the tissue RAS. Tissue RAS might be involved in tissue function of regulating tear secretion in the lacrimal gland.

Transduction, Tropism, and Biodistribution of AAV Vectors in the Lacrimal Gland

The lacrimal gland (LG) delivers defensive and metabolic factors to the ocular surface. These functions may be disrupted in several diseases, and for most of them there is no cure. The aim of this study is to investigate conditions and limitations for using adeno-associated virus (AAV) vectors as gene transfer agents to LG. Eight-week-old Balb/c mice were used to investigate route, gene expression, and time course of AAV gene vector transfer to LG. AAV vectors encoding firefly luciferase were administered to the LG and luciferase expression was evaluated in vivo by immunohistochemistry. Ocular surface and neutralizing antibodies were also evaluated. The present work revealed that AAV vectors are able to delivery DNA to the LGs of mice. Direct injection had the highest level of transduction, and topical ocular drops the lowest. Overall, the AAV strain with highest transduction activity as measured by both luminescence and immunohistochemistry was AAV9, followed by AAV 5w8 and AAV5. Transduction was not different between sexes, could be detected as soon as 24 hours after injection, and lasted for at least 30 days (study termination). No tissue damage was observed when compared with controls. All vectors with detectable LG transduction induced neutralizing antibodies. LG gene delivery by AAV vectors appears to be both safe and well tolerated. The choice of vector influences both the overall transduction activity, as well as the spread of vector to other organs. This work supports the use of AAV-mediated gene therapy for dry eye.

Polycystin-2 Expression and Function in Adult Mouse Lacrimal Acinar Cells

Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca(2+) homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca(2+) concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP(3)Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca(2+) release channels, transient receptor potential polycystin family (TRPP) channels. Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca(2+) channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system. All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues. The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology.

Pharmacological Cholinergic Blockade Stimulates Inflammatory Cytokine Production and Lymphocytic Infiltration in the Mouse Lacrimal Gland

To investigate the effects of cholinergic blockade on inflammatory cell infiltration and cytokine production in the mouse lacrimal gland (LG). C57BL/6 mice were untreated (UT) or received subcutaneous injections of either scopolamine hydrobromide (SCOP; 0.5 mg/0.2 mL) or saline (SAL) four times daily for 2 or 5 days (2D, 5D). This was followed by a 7-day rest period in separate groups. Tear volume (cotton thread) and tear epidermal growth factor (EGF, by ELISA) concentrations were measured. Extraorbital LGs were surgically excised and sectioned or lysed for gene expression analysis. Immunohistochemistry evaluated immunophenotype of infiltrating cells. Expression of EGF and T helper (Th)-1, -2, and -17-associated cytokines in LGs was evaluated by real-time PCR. Goblet cell density was evaluated in periodic acid Schiff-stained conjunctival sections. Tear volume and EGF protein levels were significantly reduced in SCOP5D mice compared with controls, indicating that cholinergic blockade decreased LG secretory function. LGs of SCOP2D and SCOP5D mice showed an increased density of CD4(+), CD11c+, CD11b+, and myeloperoxidase+ cells compared with UT controls. At day 5, these cells were significantly elevated compared with SAL-treated counterparts. Elevated levels of IL-17A, IL-17R, IFN-γ, IL-12Rβ1, IL-2, IL-13, IL-6, IL-1β, and TNF-α transcripts were noted in SCOP2D mice and IFN-γ, TGF-β1, and IL-18R transcripts in SCOP5D mice. Pharmacological blockade of lacrimal secretion induced a significant CD4(+) infiltration in the LG, mimicking Sjögren's syndrome. The mRNA expression profile revealed elevations of a mix of inflammatory cytokines and Th-1-associated factors.

Lymphocytic infiltration leads to degradation of lacrimal gland extracellular matrix structures in NOD mice exhibiting a Sjögren's syndrome-like exocrinopathy

We previously reported that lacrimal glands (LGs) of male non-obese diabetic (NOD) mice, an established mouse model of autoimmune inflammatory LG disease that displays many features of human LGs in patients afflicted with Sjögren's syndrome (SjS), exhibit significant degradation of extracellular matrix (ECM) structures as well as increased expression of matrix metalloproteinases (MMPs). The purpose of the current study was to expand the spectrum of proteases identified, to clarify their probable origin as well as to identify the contribution of these changes to disease pathogenesis. We explored in depth the changes in ECM structures and ECM protease expression at the onset of disease (6 weeks) versus late stage disease (18 weeks) in male NOD mouse LGs, relative to LGs of age-matched male NOD scid, a severely immunocompromised congenic strain, and healthy BALB/c mice. LG tissues were examined using routine histological, immunohistochemical, Western Blot and gene expression analyses novel multiphoton imaging technologies. We further characterized the profile of infiltrating immune cells under each condition using flow cytometry. Our results show that the initial infiltrating cells at 6 weeks of age are responsible for increased MMP and cathepsin H expression and therefore initiate the LG ECM degradation in NOD mice. More importantly, NOD scid mice exhibited normal LG ECM structures, indicating the lymphocytes seen in the LGs of NOD mice are responsible for the degradation of the LG ECM. The disease-related remodeling of LG ECM structures may play a crucial role in altering the acinar signaling environment, disrupting the signaling scaffolds within the cells, which are required to mobilize the exocytotic trafficking machinery, ultimately leading to a loss of LG function in patients afflicted with SjS.

Erratum: An experiment of the combined treatment of traditional Lei-huo-jiu therapy with Chinese medicine for the lacrimal gland of Sjögren's syndrome.

This experiment chooses nonobese diabetic (NOD) mouse as the animal model of Sjögren’s syndrome and investigates the morphologic changes, the expression of inflammatory factors and growth factors of this mouse’s lacrimal gland in response to a combined treatment of traditional Lei-huo-jiu therapy alone and in combination with Chinese medicine. The methods were to (1) use a morphological approach to directly observe pathological changes of the lacrimal gland in response to combined treatment and (2) to detect the level of tumor necrosis factor (TNF)-α, interleukin (IL)-1, and nuclear factor kappa B (NF-κB) in lacrimal gland tissue caused by the combined treatments using a immunohistochemical approach. There is a reduction of the mast cell’s degranulation and modulation of the level of cytokines in TNF-α, IL-1, and NF-κB in the combined therapy group. The combined treatment of traditional Lei-huo-jiu therapy with Chinese medicine can improve the pathological changes of the lacrimal gland tissue of the NOD mouse through modulating the level of TNF-α, IL-1, and NF-κB which results in improved tear secretion and function of the lacrimal gland.

Altered expression of genes functioning in lipid homeostasis is associated with lipid deposition in NOD mouse lacrimal gland

Functional atrophy and accompanying lymphocytic infiltration and destruction of the lacrimal gland (LG) are characteristics of Sjögren's Syndrome (SjS). The male NOD mouse is an experimental model for the autoimmune exocrinopathy that develops in the LG of SjS patients. Acinar cells in LG of male NOD mice aged 3–4 months were previously shown to accumulate lipid droplets. In the current study, analysis of lipid components revealed that the accumulated lipids were mostly cholesteryl esters (CE). Gene expression microarray analysis followed by real-time RT-PCR revealed alterations in the expression of several genes involved in lipid homeostasis in LG of 12-week-old male NOD mice relative to matched BALB/c controls. A series of upregulated genes including apolipoprotein E, apolipoprotein F, hepatic lipase, phosphomevalonate kinase, ATP-binding cassette D1 and ATP-binding cassette G1 were identified. Comparison of liver mRNAs to LG mRNAs in BALB/c and NOD mice revealed that the differential expressions were LG-specific. Gene expression profiles were also characterized in LGs of female mice, younger mice and immune-incompetent NOD SCID mice. Investigation of the cellular distribution of Apo-E and Apo-F proteins suggested that these proteins normally coordinate to mediate lipid efflux from the acinar cells but that dysfunction of these processes due to missorting of Apo-F may contribute to CE deposition. Finally, the initiation and extent of lipid deposition were correlated with lymphocytic infiltration in the LG of male NOD mice. We propose that impaired lipid efflux contributes to lipid deposition, an event that may contribute to the development and/or progression of dacryoadenitis in the male NOD mouse.

Purification and Characterization of Mouse Lacrimal Gland Epithelial Cells and Reconstruction of an Acinarlike Structure in Three-Dimensional Culture

To clarify which factors are involved in postnatal lacrimal gland development, the expressions of several growth factors and their receptors were analyzed in purified lacrimal gland epithelial and mesenchymal cells. Reconstruction of acinarlike structures in three-dimensional culture conditions from this epithelial cell population was attempted. Lacrimal gland epithelial and mesenchymal cells were isolated from newborn mice and purified using nylon mesh and collagenase. By real-time reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blotting, the expressions of several growth factors and their receptors were analyzed. Responses of epithelial cells to the growth factors were analyzed by the addition of these factors to the culture medium. Fibroblast growth factor (FGF)10 and hepatocyte growth factor (HGF) were more intensely expressed in mesenchymal cells than in epithelial cells, and their receptors (FGFR2IIIb and c-Met, respectively) were less intensely expressed, whereas the expression of epidermal growth factor (EGF) and its receptor showed no significant difference between cell types. In the monolayer culture, cell viability was activated by the addition of EGF or HGF to epithelial cells, but no response was observed when FGF10 was added. The epithelial cells formed clusters with lumina when cultured on basement membrane matrix. These clusters contained secretion granules and showed positive immunostaining for aquaporin-5. EGF and HGF were considered to act in an autocrine/paracrine manner in and around the postnatal lacrimal gland, whereas epithelial cells did not respond to FGF10. It was suggested that certain extracellular matrix conditions accommodate these epithelial cells to reconstruct functional acinarlike structures.

Altered Distribution of Aquaporin 5 and its C-Terminal Binding Protein in the Lacrimal Glands of a Mouse Model for Sjögren's Syndrome

Purpose: To investigate the distribution and expression of aquaporin 5 (AQP5) and its C-terminal binding protein in the apical membrane of the lacrimal glands (LGs) in a mouse model for Sjögren's syndrome (SS). Methods: The LGs of NOD mice (mouse model for SS) and ICR mice (normal control) were homogenized and delivered into the affinity columns bound to synthetic AQP5 C-terminal peptide. The eluates were analyzed by electrophoresis and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) techniques.Results: AQP5 from the NOD mice exhibited the capacity to bind a 21-kDa protein that was lacking in the ICR mice. Instead, ICR mouse expressed a 17-kDa AQP5 binding protein that was absent in LGs of SS. LC-MS/MS analysis revealed these respective proteins to be major urinary protein 4 (MUP4) and prolactin-inducible protein (PIP). The treatment of ICR mice with antisense PIP oligonucleotides decreased immunostaining of AQP5 in the apical membrane. Conclusions: These observations suggest that the binding of PIP to the C-terminal portion of AQP5 may cause AQP5 to be transported to the apical membrane of LGs. Correction of the aberrant binding of PIP to the AQP5 C-terminus could normalize AQP5 trafficking to the apical membrane, leading to a treatment for patients with SS.

Identification and Functional Distribution of Intracellular Ca2+ Channels in Mouse Lacrimal Gland Acinar Cells

We have determined the presence and cellular distribution of intracellular calcium channels, inositol 1, 4, 5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in adult and postnatal (P10) lacrimal gland acinar cells. Western blot analysis of both P10 cultures and adult tissue identified the presence of each IP3R and RyR isotypes. The immunocytochemistry analysis showed a differential cellular distribution of these calcium channels where the nuclear envelope, endoplasmic reticulum (ER) and Golgi apparatus membranes represent areas with highest levels of channel expression. This IP3R and RyR isotype distribution is confirmed by the immuno-EM results. The findings described in this study are in agreement with published pharmacological data that shows the participation of these channels in the secretion process of the lacrimal gland acinar cells. Furthermore, the differential subcellular distribution between the isoforms could indicate a potential role of these intracellular Ca2+ channels on the regulation of specific cellular functions.

The α1- and β1-Adrenergic Modulation of Lacrimal Gland Function in the Mouse

To determine the expression patterns of alpha(1)- and beta(1)-adrenergic receptors in the mouse exorbital lacrimal gland (LG). An alpha- and beta-receptor agonist and antagonist were used to elucidate the receptors' relevance to protein secretion. Mouse LGs were processed for single- and double-labeled indirect immunofluorescence studies and examined with confocal scanning microscopy. Protein secretion was measured from gland fragments in response to adrenergic agonists. Extensive alpha(1)-immunoreactivity (IR) was found on the surface and cytoplasm of acinar cells and much more alpha(1)-IR in the interstitial areas. In contrast, more beta(1)-IR was found in the LG, and most beta(1)-IR appeared to concentrate in the cytoplasm of acinar cells, with almost no beta(1)-IR in the interstitial areas. The protein secretion in response to phenylephrine and isoproterenol showed that direct stimulation of either the alpha(1)- or beta(1)-receptor could induce significant protein secretion from LGs. The specificity of this stimulation was further indicated by the effects of adrenergic antagonists. No synergism was observed between alpha(1)- and beta-receptor-mediated protein secretions. The results support the notion that there is extensive adrenergic control in the mouse LG. The adrenergic receptors may be a better choice of markers, compared with tyrosine hydroxylase and dopamine beta-hydroxylase, to reflect the extent of adrenergic control because circulating norepinephrine in the bloodstream should be taken into consideration. Both confocal microscopy observations and protein secretion data suggest the presence of alpha(1)- and beta(1)-mediated pathways in the mouse LG.

A single injection of interleukin-1 induces reversible aqueous-tear deficiency, lacrimal gland inflammation, and acinar and ductal cell proliferation

Emerging studies from our laboratory demonstrate that interleukin-1 (IL-1) family members play a major role in impairing lacrimal gland functions. Here we have extended our investigations to observe the effects of IL-1 on aqueous tear production, lacrimal gland secretion, lacrimal gland histology, and acinar and ductal cell proliferation. We demonstrate that a single injection of IL-1 into the lacrimal glands inhibited neurally- as well as agonist-induced protein secretion resulting in decreased tear output. Meanwhile, IL-1 injection induced a severe, but reversible (7–13 days), inflammatory response that led to destruction of lacrimal gland acinar epithelial cells. Finally, we demonstrate that as the inflammatory response subsided and lacrimal gland secretion and tear production returned to normal levels, there was increased proliferation of acinar and ductal epithelial cells. Our work uncovers novel effects of IL-1 on lacrimal gland functions and the potential regenerative capacity of the mouse lacrimal gland.

Altered phosphorylation of cytoskeletal proteins in mutant protein phosphatase 2A transgenic mice

Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes with diverse functions under physiologic and pathologic conditions such as Alzheimer’s disease. All PP2A holoenzymes have in common a catalytic subunit C and a structural scaffolding subunit A. These core subunits assemble with various regulatory B subunits to form heterotrimers with distinct functions in the cell. Substrate specificity of PP2A in vitro is determined by regulatory subunits with leucine 309 of the catalytic subunit C playing a crucial role in the recruitment of regulatory subunits into the complex. Here we expressed a mutant form of Cα, L309A, in brain and Harderian (lacrimal) gland of transgenic mice. We found an altered recruitment of regulatory subunits into the complex, demonstrating a role for the carboxyterminal leucine of Cα in regulating holoenzyme assembly in vivo. This was associated with an increased phosphorylation of tau in brain and an impaired dephosphorylation of vimentin demonstrating that both cytoskeletal proteins are in vivo substrates of distinct PP2A holoenzyme complexes.

β‐Adrenergic Stimulation Increases Protein Secretion But Not Fluid Flow in Mouse Lacrimal Gland

Activation of either α- and β-adrenergic receptors is known to induce protein secretion from isolated acinar cells and gland fragments. Here, we determined if β-adrenergic receptor activation with isoproterenol (ISO) stimulates fluid secretion. In anesthetized C57 mice, fluid flow from the lacrimal duct was measured in calibrated microcapillaries via videomicroscopy; secretion was elicited by adding 10 μM carbachol (CARB) or ISO to the fluid bathing the gland. Acinar cells were isolated and plated on matrigel. After 24 h, cells were exposed to CARB or ISO and changes in cell volume and membrane currents were measured via videomicroscopy and patch-clamping. In intact glands, CARB elicited a prompt, reversible rise in tear flow, but ISO had no detectable effect. In isolated cells, CARB induced a ~10% shrinkage, consistent with the onset of fluid secretion. ISO had no effect on cell volume in ~80% of cells studied (n=26 total); ~20% showed marked shape changes and a variable degree of shrinkage that may be associated with protein secretion. CARB resulted in marked increases in outward and inward membrane currents consistent with activation of K+ and Cl− channels; the currents were much lower with ISO and ½ of the cells did not show any response, despite normal resting potential and currents. Conclusion β-adrenergic does not stimulate lacrimal fluid secretion. This result supports the concept that there is differential regulation of water and protein secretion in lacrimal gland. Funded by NIH EY 14604

The role of the forkhead transcription factor, Foxc1, in the development of the mouse lacrimal gland

The lacrimal gland produces secretions that lubricate and protect the cornea of the eye. Foxc1 encodes a forkhead/winged helix transcription factor required for the development of many embryonic organs. Autosomal dominant mutations in human FOXC1 cause eye disorders such as Axenfeld-Rieger Syndrome and glaucoma iris hypoplasia, resulting from malformation of the anterior segment of the eye. We show here that lacrimal gland development is severely impaired in homozygous null Foxc1 mouse mutants, with reduced outgrowth and branching. Foxc1 is expressed in both the epithelium of the lacrimal gland and the surrounding mesenchyme. FGF10 stimulates the growth and branching morphogenesis in cultures of wild type and Foxc1 mutant gland epithelial buds. However, using micromass culture of lacrimal gland mesenchyme, we show that Bmp7 induces wild type mesenchyme cells to aggregate, but Foxc1 mutant cells do not respond. This study demonstrates that Foxc1 mediates the BMP signaling required for lacrimal gland development.

Estrogen’s and Progesterone’s Impact on Gene Expression in the Mouse Lacrimal Gland

The hypothesis tested in the study was that the effect of estrogen and progesterone on the lacrimal gland is mediated through specific receptors and that hormonal effects involve the regulation of gene expression and protein synthesis. Lacrimal glands were collected from young adult, ovariectomized mice, that were treated with 17beta-estradiol, progesterone, 17beta-estradiol plus progesterone or vehicle for 2 weeks. Glands were pooled according to treatment, processed for the isolation of RNA, and evaluated for differentially expressed mRNAs by using gene microarrays. Bioarray data were analyzed with sophisticated bioinformatics and statistical programs. The expression of selected genes was verified by using gene chips and quantitative real-time PCR methods. The results demonstrate that 17beta-estradiol, progesterone, or both hormones together significantly influences the expression of hundreds of genes in the mouse lacrimal gland. Sex steroid treatment led to numerous alterations in gene activities related to transcriptional control, cell growth and/or maintenance, cell communication, signal transduction, enzyme catalysis, immune expression, and the binding and metabolism of nucleic acids and proteins. A number of the 17beta-estradiol, progesterone or 17beta-estradiol plus progesterone effects on gene expression were similar, but most were unique to each treatment. Of particular interest was the finding that these hormones seem to contribute little to the known sex-related differences in gene expression of the lacrimal gland. These results support the hypothesis that estrogen's and progesterone's action on the lacrimal gland involves the regulation of numerous genes. However, these hormone effects do not appear to represent a major factor underlying the sexual dimorphism of gene expression in lacrimal tissue.

Androgen regulation of gene expression in the mouse lacrimal gland

The objective of this study was to determine the nature and extent of androgen influence on gene expression in the lacrimal gland. Lacrimal glands were obtained from orchiectomized mice that had been treated with testosterone or vehicle for 2 weeks, as well as from testicular feminized mice and their Tabby controls. Samples were pooled according to experiment, processed for the isolation of RNA, and analyzed for differentially expressed mRNAs by using primarily CodeLink Bioarrays, GEM 1 and 2 gene chips and quantitative real-time PCR (qPCR) procedures. Gene chip data were analyzed with GeneSifter.Net software. Our results demonstrate that testosterone regulates the expression of over 2000 genes in the lacrimal gland. Gene ontologies most affected by androgen treatment included those related to cell growth, proliferation and metabolism, cell communication and transport, nucleic acid binding, signal transduction and receptor activities. Our findings also indicate that androgen action may be mediated, at least in part, through classical androgen receptors, and may contribute to the sex-related differences in gene expression of lacrimal tissue. Overall, these results support our working hypothesis that androgen action on the lacrimal gland is mediated primarily through a receptor-associated regulation of gene transcription.