Drosophila TAB2 is required for the immune activation of JNK and NF-kappaB

The TAK1 plays a pivotal role in the innate immune response of Drosophila by controlling the activation of JNK and NF-kappaB. Activation of TAK1 in mammals is mediated by two TAK1-binding proteins, TAB1 and TAB2, but the role of the TAB proteins in the immune response of Drosophila has not yet been established. Here, we report the identification of a TAB2-like protein in Drosophila called dTAB2. dTAB2 can interact with dTAK1, and stimulate the activation of the JNK and NF-kB signaling pathway. Furthermore, we have found that silencing of dTAB2 expression by dsRNAi inhibits JNK activation by peptidoglycans (PGN), but not by NaCl or sorbitol. In addition, suppression of dTAB2 blocked PGN-induced expression of antibacterial peptide genes, a function normally mediated by the activation of NF-kappaB signaling pathway. No significant effect on p38 activation by dTAB2 was found. These results suggest that dTAB2 is specifically required for PGN-induced activation of JNK and NF-kappaB signaling pathways.     

Okadaic acid suppresses TPA-induced differentiation by stimulating G1/S transition in human myeloblastic leukaemia ML-1 cells

The association between the phosphorylation status of the retinoblastoma protein, pRb and changes in cell cycle control caused by either protein kinase C (PKC) or protein kinase A (PKA) stimulation was evaluated in human myeloblastic leukaemia ML-1 cells. TPA-induced PKC activation resulted in dephosphorylation of pRb and subsequently induced ML-1 differentiation based on morphological changes and CD14 expression. In the present study, we showed that inhibition of protein phosphatases (PP-1 and PP-2a) prevented the TPA-induced differentiation in ML-1 cells. Preinhibition of PP-1 and PP-2a activities with 1–100 nM okadaic acid dose-dependently blunted the decrease in the phosphorylation status of pRb obtained with TPA and overrode cell cycle arrest. PKA stimulation with 8-chlorophenylthio-cAMP (100 µM) decreased cell proliferation by 65% and the distribution of cells in the G₁ phase significantly increased from 38% to 83% concomitant with a 34% decline in the number of cells present in the S phase. In addition, PKA stimulation significantly decreased the pRb phosphorylation status but did not elicit CD14 expression, indicating that cAMP-induced dephosphorylation of pRb cannot by itself trigger differentiation in ML-1 cells.     

Adenomatous Polyposis Coli Mutation Leads to Myopia Development in Mice

Myopia incidence in China is rapidly becoming a very serious sight compromising problem in a large segment of the general population. Therefore, delineating the underlying mechanisms leading to myopia will markedly lessen the likelihood of other sight compromising complications. In this regard, there is some evidence that patients afflicted with familial adenomatous polyposis (FAP), havean adenomatous polyposis coli (APC) mutation and a higher incidence of myopia. To clarify this possible association, we determined whether the changes in pertinent biometric and biochemical parameters underlying postnatal refractive error development in APC^Min mice are relevant for gaining insight into the pathogenesis of this disease in humans. The refraction and biometrics in APC^Min mice and age-matched wild-type (WT) littermates between postnatal days P28 and P84 were examined with eccentric infrared photorefraction (EIR) and customized optical coherence tomography (OCT). Compared with WT littermates, the APC^Min mutated mice developed myopia (average -4.64 D) on P84 which was associated with increased vitreous chamber depth (VCD). Furthermore, retinal and scleral changes appear in these mice along with: 1) axial length shortening; 2) increased retinal cell proliferation; 3) and decreased tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of DA synthesis. Scleral collagen fibril diameters became heterogeneous and irregularly organized in the APC^Min mice. Western blot analysis showed that scleral alpha-1 type I collagen (col1α1) expression also decreased whereas MMP2 and MMP9 mRNA expression was invariant. These results indicate that defective APC gene function promotes refractive error development. By characterizing in APC^Min mice ocular developmental changes, this approach provides novel insight into underlying pathophysiological mechanisms contributing to human myopia development.     

Comparison of Diagnostic Tests in Distinct Well-Defined Conditions Related to Dry Eye Disease

Purpose

This study compares signs, symptoms and predictive tools used to diagnose dry eye disease (DED) and ocular surface disorders in six systemic well-defined and non-overlapping diseases. It is well known that these tests are problematic because of a lack of agreement between them in identifying these conditions. Accordingly, we provide here a comparative clinical profile analysis of these different diseases.

Methods

A spontaneous and continuous sample of patients with Sjögren''s syndrome (SS) (n = 27), graft-versus-host-disease (GVHD) (n = 28), Graves orbitopathy (n = 28), facial palsy (n = 8), diabetes mellitus without proliferative retinopathy (n = 14) and glaucoma who chronically received topical drugs preserved with benzalkonium chloride (n = 20) were enrolled. Evaluation consisted of a comprehensive protocol encompassing: (1) structured questionnaire - Ocular Surface Disease Index (OSDI); (2) tear osmolarity (TearLab Osmolarity System - Ocusense); (3) tear film break-up time (TBUT); (4) fluorescein and lissamine green staining; (5) Schirmer test and (6) severity grading.

Results

One hundred and twenty five patients (aged 48.8 years-old±14.1, male:female ratio = 0.4) were enrolled in the study, along with 24 age and gender matched controls. Higher scores on DED tests were obtained in Sjögren Syndrome (P<0.05), except for tear film osmolarity that was higher in diabetics (P<0.001) and fluorescein staining, that was higher in facial palsy (P<0.001). TFBUT and OSDI correlated better with other tests. The best combination of diagnostic tests for DED was OSDI, TBUT and Schirmer test (sensitivity 100%, specificity 95% and accuracy 99.3%).

Conclusions

DED diagnostic test results present a broad range of variability among different conditions. Vital stainings and TBUT correlated best with one another whereas the best test combination to detect DED was: OSDI/TBUT/Schirmer.     

TRPV1 Potentiates TGFβ-Induction of Corneal Myofibroblast Development through an Oxidative Stress-Mediated p38-SMAD2 Signaling Loop

Injuring mouse corneas with alkali causes myofibroblast expression leading to tissue opacification. However, in transient receptor potential vanilloid 1 channel (TRPV1-/-) knockout mice healing results in transparency restoration. Since TGFβ is the primary inducer of the myofibroblast phenotype, we examined the mechanism by which TRPV1 affects TGFβ-induced myofibroblast development. Experiments were performed in pig corneas and human corneal fibroblasts (HCFs). Immunohistochemical staining of α-smooth muscle actin (α-SMA) stress fibers was used to visualize myofibroblasts. Protein and phosphoprotein were determined by Western blotting. siRNA transfection silenced TRPV1 gene expression. Flow cytometry with a reactive oxygen species (ROS) reporting dye analyzed intracellular ROS. [Ca2+]I was measured by loading HCF with fura2. In organ cultured corneas, the TRPV1 antagonist capsazepine drastically reduced by 75% wound-induced myofibroblast development. In HCF cell culture, TGF-β1 elicited rapid increases in Ca2+ influx, phosphorylation of SMAD2 and MAPKs (ERK1/2, JNK1/2 and p38), ROS generation and, after 72 hrs myofibroblast development. SMAD2 and p38 activation continued for more than 16 h, whereas p-ERK1/2 and p-JNK1/2 waned within 90 min. The long-lived SMAD2 activation was dependent on activated p38 and vice versa, and it was essential to generate a > 13-fold increase in α-SMA protein and a fully developed myofibroblast phenotype. These later changes were markedly reduced by inhibition of TRPV1 or reduction of the ROS generation rate. Taken together our results indicate that in corneal derived fibroblasts, TGFβ- induced myofibroblast development is highly dependent on a positive feedback loop where p-SMAD2-induced ROS activates TRPV1, TRPV1 causes activation of p38, the latter in turn further enhances the activation of SMAD2 to establish a recurrent loop that greatly extends the residency of the activated state of SMAD2 that drives myofibroblast development.     

Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells

TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4alpha-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca(2+) ([Ca(2+)](i)) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca(2+)](i) transients. Similarly, exposure to either ruthenium red or Ca(2+)-free Ringer's solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca(2+)-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca(2+)](i) transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.