Mice lacking inositol 1,4,5-trisphosphate receptors exhibit dry eye.

Tear secretion is important as it supplies water to the ocular surface and keeps eyes moist. Both the parasympathetic and sympathetic pathways contribute to tear secretion. Although intracellular Ca2+ elevation in the acinar cells of lacrimal glands is a crucial event for tear secretion in both the pathways, the Ca2+ channel, which is responsible for the Ca2+ elevation in the sympathetic pathway, has not been sufficiently analyzed. In this study, we examined tear secretion in mice lacking the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3 (Itpr2-/-;Itpr3-/-double-knockout mice). We found that tear secretion in both the parasympathetic and sympathetic pathways was abolished in Itpr2-/-;Itpr3-/- mice. Intracellular Ca2+ elevation in lacrimal acinar cells after acetylcholine and epinephrine stimulation was abolished in Itpr2-/-;Itpr3-/- mice. Consequently, Itpr2-/-;Itpr3-/- mice exhibited keratoconjunctival alteration and corneal epithelial barrier disruption. Inflammatory cell infiltration into the lacrimal glands and elevation of serum autoantibodies, a representative marker for Sjögren's syndrome (SS) in humans, were also detected in older Itpr2-/-;Itpr3-/- mice. These results suggested that IP3Rs are essential for tear secretion in both parasympathetic and sympathetic pathways and that Itpr2-/-;Itpr3-/- mice could be a new dry eye mouse model with symptoms that mimic those of SS.

The effect of topical amiloride eye drops on tear quantity in rabbits.

PURPOSE: To investigate the presence of epithelial sodium channels (ENaC) in rabbit and human conjunctival epithelium and to test the effects of topical amiloride, a potassium-sparing diuretic that blocks the ENaC, on tear quantity in rabbits. METHODS: Both healthy normal human and rabbit conjunctival tissues underwent immunohistochemistry staining for ENaC-α and γ subunits as well as for reverse transcription-polymerase chain reaction (RT-PCR) for detection of ENaC-α and ENaC-γ subunit mRNA expression. Rabbits were instilled topical amiloride eye drops and tear function tests were performed before and after instillations. RESULTS: Immunohistochemical staining for ENaC-α subunit in all rabbit eyes showed positive staining in apical and basal conjunctival epithelial cells. Human conjunctival epithelia revealed positive staining with ENaC-α antibody especially in the apical and basal layers. Immunohistochemistry staining with ENaC- γ antibody also revealed positive staining of the conjunctival epithelial cells especially in the basal layers. The ENaC-α mRNA was detected in samples from healthy white rabbit conjunctival epithelia, and ENaC-α and ENaC- γ mRNAs were detected in samples from healthy human conjunctival epithelia. The mean tear quantity showed a significant increase at 15 and 30 min compared to the pre-instillation value in eyes assigned to amiloride eye drops. The mean tear quantity at 15 and 30 min was significantly higher in the amiloride group compared to the control eyes. CONCLUSIONS: Topical amiloride application appears to increase the quantity of preocular tears owing to inhibition of conjunctival epithelial sodium channels.

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 Sjogren'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.

The difference of aquaporin 5 distribution in acinar and ductal cells in lacrimal and parotid glands.

PURPOSE: This study was designed to clarify the physiological function and tissue distribution of aquaporin 5 (AQP5) in the lacrimal and parotid glands. METHODS: Saliva and tear volumes were compared in AQP5 knockout (AQP5-/-) mice and wild-type mice. Immunohistochemistry and immunoblot analysis were performed in wild-type and AQP5-/- mice. RESULTS: Immunofluorescence of AQP5 staining showed that AQP5 was localized mainly in the ductal cells rather than in the acinar cells of the lacrimal gland. In contrast, in the parotid gland, AQP5 was observed abundantly in acinar cells with undetectable staining in ductal cells. Tear secretion was not changed in AQP5-/- mouse, although saliva secretion was significantly reduced. CONCLUSIONS: AQP5 distribution in acinar cells and ductal cells was completely opposite in the lacrimal and parotid glands. The physiological role of AQP5 might be dependent on the characteristic tissue distribution of the protein in the lacrimal and parotid glands.

Down-regulation of Fas-ligand mRNA in Sjögren's syndrome patients with enlarged exocrine glands.

We have reported that Sjögren's syndrome (SS) patients with enlarged exocrine glands (EEG) formerly referred to as Mikulicz's disease were defective with Fas-ligand (FasL) expression in PBL and lacrimal glands (LGs). To investigate the mechanisms of reduced FasL expression in SS patients with EEG, FasL mRNA expression level was determined using real-time PCR. The FasL gene promoter region (from - 1197 to - 3) was also amplified using PCR and specific primers. Expression of the FasL mRNA in the LGs and PBLs of three SS patients with EEG was significantly decreased. Direct sequencing revealed a heterozygous point mutation ( - 259T/C) in the FasL gene promoter region in one SS patient with EEG. A luminescent beta-galactosidase (beta-gal) reporter assay using a pbetagal Enhancer Vector demonstrated that beta-gal activity from the vector including the mutant ( - 259C) FasL (pbetagal/mFasL) gene promoter region (735 +/- 42) was similar (p = 0.13) to that from a pbetagal Enhancer Vector without the gene promoter region (603 +/- 66). On the other hand, the beta-gal activity was significantly lower (p < 0.0001) than that from a vector including the wild-type ( - 259T) FasL (pbetagal/wFasL) (3226 +/- 148). In conclusion, the down-regulation of FasL in SS patients with EEG may be due to transcriptional regulation, and the point mutation at - 259T/C in the FasL gene promoter region may lead to the down-regulation of FasL mRNA expression and the lymphoproliferative process observed in SS patients with EEG.

Nuclear receptor mediated gene regulation through chromatin remodeling and histone modifications.

Nuclear steroid/thyroid vitamin A/D receptor genes form a gene superfamily and encode DNA-binding transcription factors that control the transcription of target genes in a ligand-dependent manner. It has become clear that chromatin remodeling and the modification of histones, the main components of chromatin, play crucial roles in gene transcription, and many distinct classes of NR-interacting co-regulators have been identified that perform significant roles in gene transcription. Since NR dysfunction can lead to the onset or progression of endocrine disease, elucidation of the mechanisms of gene regulation mediated by NRs, as well as the identification and characterization of co-regulator complexes (especially chromatin remodeling and histone-modifying complexes), is essential not only for better understanding of NR ligand function, but also for pathophysiological studies and the development of therapeutic interventions in humans.

Ligand-induced transrepression by VDR through association of WSTF with acetylated histones.

We have previously shown that the novel ATP-dependent chromatin-remodeling complex WINAC is required for the ligand-bound vitamin D receptor (VDR)-mediated transrepression of the 25(OH)D3 1alpha-hydroxylase (1alpha(OH)ase) gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand-induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone-interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant-negative mutant in terms of ligand-induced transrepression of the 1alpha(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated his tones, which appears to be indispensable for VDR/promoter association for ligand-induced transrepression of 1alpha(OH)ase gene expression.

Novel gender-related regulation of CYP2C12 gene expression in rats.

The expression of CYP2C12 by GH occurs in female but not in male rat livers. Direct injection of the CYP2C12 promoter-luciferase gene into male rat livers showed that the CYP2C12 promoter was active in both male and female rats. Thus, to further examine one or more factors that regulate the gender-related expression of CYP2C12, male rats were treated with trichostatin A, a specific inhibitor of histone deacetylase capable of condensing the chromatin structure. Interestingly, the expression of CYP2C12 by GH was seen even in the livers of male rats, indicating that histone deacetylase contributes to the suppression of CYP2C12 expression in male rats. Deoxyribonuclease I hypersensitive assay using nuclei from the livers of male or female rats revealed that the chromatin structure of the CYP2C12 gene was gender specific: a hypersensitive site at a position -4.2 kb containing GH-responsive element that bound to signal transducer and activator of transcription 5 (STAT5), termed as HS (hypersensitive site) 1, was specific for female rat livers, whereas a hypersensitive site at a position -3 kb, designated as HSm (male-specific hypersensitive site), was characteristic of male rat livers. A -3425/-3275 region within HSm functioned as a negative regulatory region, when the region was inserted in front of simian virus 40 promoter. Gel shift assay demonstrated that both CCAAT/enhancer-binding protein alpha and beta bound to the -3425/-3275 region. Based on these results, we conclude that the gender-related expression of the CYP2C12 gene results from the inaccessibility of to STAT5 to the GH-responsive element by chromatin condensation seen in male rat livers, and from the presence of the male-specific HSm that acts as a silencer.