GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 1

Identification of MFRP and the secreted serine proteases PRSS56 and ADAMTS19 as part of a molecular network involved in ocular growth regulation.

Precise regulation of ocular size is a critical determinant of normal visual acuity. Although it is generally accepted that ocular growth relies on a cascade of signaling events transmitted from the retina to the sclera, the factors and mechanism(s) involved are poorly understood. Recent studies have highlighted the importance of the retinal secreted serine protease PRSS56 and transmembrane glycoprotein MFRP, a factor predominantly expressed in the retinal pigment epithelium (RPE), in ocular size determination. Mutations in PRSS56 and MFRP constitute a major cause of nanophthalmos, a condition characterized by severe reduction in ocular axial length/extreme hyperopia. Interestingly, common variants of these genes have been implicated in myopia, a condition associated with ocular elongation. Consistent with these findings, mice with loss of function mutation in PRSS56 or MFRP exhibit a reduction in ocular axial length. However, the molecular network and cellular processes involved in PRSS56- and MFRP-mediated ocular axial growth remain elusive. Here, we show that Adamts19 expression is significantly upregulated in the retina of mice lacking either Prss56 or Mfrp. Importantly, using genetic mouse models, we demonstrate that while ADAMTS19 is not required for ocular growth during normal development, its inactivation exacerbates ocular axial length reduction in Prss56 and Mfrp mutant mice. These results suggest that the upregulation of retinal Adamts19 is part of an adaptive molecular response to counteract impaired ocular growth. Using a complementary genetic approach, we show that loss of PRSS56 or MFRP function prevents excessive ocular axial growth in a mouse model of early-onset myopia caused by a null mutation in Irbp, thus, demonstrating that PRSS56 and MFRP are also required for pathological ocular elongation. Collectively, our findings provide new insights into the molecular network involved in ocular axial growth and support a role for molecular crosstalk between the retina and RPE involved in refractive development

Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

<div><p>Hemoglobin (Hb) is a major protein involved in transport of oxygen (O₂). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish <i>in-vitro</i> culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-<i>κ</i>B to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11–7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-<i>κ</i>B to Hb-α promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-α and Hb-β and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-<i>κ</i>B regulatory sequences in Hb-α promoter, whose activation is associated with immune response of hPVECs, indicating Hb-α and Hb-β may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.</p></div

qPCR analysis of <i>Hb-α</i> and <i>Hb-β</i> expression in hPVECs and VK2/E6E7 cells before and after induction with LPS.

<p>Cells seeded at a density of 10⁶/well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of <i>Hb-α</i> and <i>Hb-β</i> was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p< 0.001 Vs Untreated).</p

Immunofluorescence localization of cytokeratin-13.

<p>Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).</p

Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells.

<p>Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).</p

Expression of NF-<i>κ</i>B in hPVECs and VK2/E6E7 cells.

<p>(A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10⁶/well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p<0.001 over Bay 11–7082 treated cells). (B). Western blot analysis of p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (i, iii) and VK2/E6E7 cells (ii, iv)1: Untreated cells, 2: LPS induced cells, 3: Bay 11–7082 treated cells, 4: Bay 11–7082 treated cells induced with LPS. Results were normalized to the β-actin (iii, iv), which is constitutively expressed in cells and serves as an internal standard. The blots shown are the representative pictures from three independent experiments. (C). Densitometric analysis of bands from western blots of p65-NF-κB reported in Fig 9B. The levels of p65-NF-κB were up-regulated in LPS-induced cells, whereas Bay 11–7082 repressed p65-NF-κB levels.</p

<i>Hb-α</i> and <i>Hb-β</i> expression in hPVECs and VK2/E6E7 cells.

<p>(A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, <i>Gapdh</i> (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.</p

Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS.

<p>EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.</p

Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells.

<p>hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).</p