We show that, in the malaria vector Anopheles gambiae, expression of Cecropin 1 is regulated by REL2, an NF-B-like transcription factor orthologous to Drosophila Relish. Through alternative splicing, REL2 produces a full-length (REL2-F) and a shorter (REL2-S) protein isoform lacking the inhibitory ankyrin repeats and death domain. RNA interference experiments show that, in contrast to Drosophila Relish, which responds solely to Gram-negative bacteria, the Anopheles REL2-F and REL2-S isoforms are involved in defense against the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli bacteria, respectively. REL2-F also regulates the intensity of mosquito infection with the malaria parasite, Plasmodium berghei. The adaptor IMD shares the same activities as REL2-F. Microarray analysis identified 10 additional genes regulated by REL2, including CEC3, GAM1, and LRIM1.innate immunity ͉ NF-B ͉ Relish ͉ Cecropin ͉ Imd
Summary We investigated miRNA expression changes associated with stress-induced premature senescence (SIPS) in primary cultures of human diploid fibroblasts (HDF) and human trabecular meshwork (HTM) cells. Twenty-five miRNAs were identified by miRNA microarray analysis and their changes in expression were validated by TaqMan realtime RT-PCR in three independent cell lines of HTM and HDF. SIPS in both HTM and HDF cell types was associated with significant down-regulation of four members of the miR-15 family and five miRNAs of the miR-106b family located in the oncogenic clusters miR-17–92, miR-106a-363, and miR-106b-25. SIPS was also associated with up-regulation of two miRNAs (182 and 183) from the miR-183-96-182 cluster. Transfection with miR-106a agomir inhibited the up-regulation of p21CDKN1A associated with SIPS while transfection with miR-106a antagomir led to increased p21CDKN1A expression in senescent cells. In addition, we identified retinoic acid receptor gamma (RARG) as a target of miR-182 and showed that this protein was down-regulated during SIPS in HDF and HTM cells. These results suggest that changes in miRNA expression might contribute to phenotypic alterations of senescent cells by modulating the expression of key regulatory proteins such as p21CDKN1A as well as by targeting genes that are down-regulated in senescent cells such as RARG.
The mechanisms responsible for the progressive malfunction of the trabecular meshwork (TM)-Schlemm's canal (SC) conventional outflow pathway tissue in primary open angle glaucoma (POAG) are still not fully understood. To determine whether POAG is characterized by an accumulation of senescent cells, similar to what has been described in other diseases, we have compared the levels of the senescence marker senescence-associated-β-galactosidase (SA-β-gal) in the outflow pathway cells of POAG and age-matched control donors. POAG donors demonstrated a statistically significant fourfold increase in the percentage of SA-β-gal positive cells. These results suggest a potential role for cellular senescence in the pathophysiology of the outflow pathway.
Elevated intraocular pressure (IOP) constitutes the best characterized risk for primary open-angle glaucoma (POAG). Elevated IOP is believed to result from an increase in aqueous humor outflow resistance at the level of the trabecular meshwork (TM)/ Schlemm's canal (SC). Malfunction of the TM in POAG is associated with the expression of markers for inflammation, cellular senescence, oxidative damage, and decreased cellularity. Current POAG treatments rely on lowering IOP, but there is no therapeutic approach available to delay the loss of function of the TM in POAG patients. We evaluated the effects of chronic administration of the dietary supplement resveratrol on the expression of markers for inflammation, oxidative damage, and cellular senescence in primary TM cells subjected to chronic oxidative stress (40% O 2 ). Resveratrol treatment effectively prevented increased production of intracellular reactive oxygen species (iROS) and inflammatory markers (IL1α, IL6, IL8, and ELAM-1), and reduced expression of the senescence markers sa-β-gal, lipofuscin, and accumulation of carbonylated proteins. Furthermore, resveratrol exerted antiapoptotic effects that were not associated with a decrease in cell proliferation. These results suggest that resveratrol could potentially have a role in preventing the TM tissue abnormalities observed in POAG.
MiR-204 potentially plays an important role in the regulation of multiple functions in HTM cells including apoptosis, accumulation of damaged proteins, ER stress response, and expression of inflammatory mediators.
MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H 2 O 2 . To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin 1 (ITGB1) and kinesin 2␣ (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3-untranslated region (3-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3-UTR. Targeting MiR-1832 is predominantly expressed in ciliated ectodermal cells and tissues including retina and hair cells in the organ of Corti and has highly conserved orthologs in both deuterostomes and protostomes. MiR-183 has been found to be up-regulated in the retina of a mouse model of retinitis pigmentosa (1) as well as in colorectal cancer (2-4). Despite its up-regulation in colorectal carcinoma cells, it has been proposed that miR-183 may inhibit the invasiveness of certain cancer cells (5). This potential anti-metastatic role of miR-183 is supported by the observations that miR-183 expression is inversely correlated with the metastatic potential of lung cancer cells. Its ectopic expression in highly metastatic cells can inhibit cell migration and invasion. Furthermore, the expression of the VIL2 coding protein Ezrin, which is known to be functionally important in cancer progression, has been demonstrated to be post-transcriptionally regulated by miR-183 (5).Based on the pattern of tissue expression of miR-183 it has been hypothesized that this miRNA may play some role in the development and function of ciliated neurosensory organs. Specifically, it has been proposed that miR-183 could contribute to reinforcing the post-mitotic differentiated state of hair cells (6). However, functional data for this miRNA is currently limited to the work by Wang et al. (5) on lung cancer cells.We have recently found that, although miR-183 is normally expressed only at low levels in human trabecular meshwork (HTM) cells and human diploid fibroblasts (HDF), its expression increased significantly after the induction of cellular senescence by exposure to H 2 O 2 in these two different cell types (7). Because cellular senescence is recognized as an anticancer mechanism (8), this observation has led us to hypothesize that miR-183 could play a role in the phenotypic changes characteristic of senescent cells, and, in particular, those involved ...
The results indicate that chronic exposure of TM cells to oxidative stress causes the accumulation of nondegradable material within the lysosomal compartment, leading to diminished lysosomal activity. Since the lysosomal system is responsible for the continuous turnover of cellular organelles, impaired lysosomal activity may lead to progressive failure of cellular TM function with age.
Malaria infection results in increased expression of immune responsive genes, including those encoding antimicrobial peptides such as Gambicin (Gam1) and Cecropin A (Cec1). Understanding how these genes are regulated will provide insights how the mosquito immune system is activated by Plasmodium. We previously have shown that Cec1 was primarily regulated by the Imd-Relish (REL2) pathway in the Anopheles gambiae Sua1B cell line. We show here that expression of Defensin A (Def1) and Gam1 was reduced after RNA interference against components of the Imd-REL2 pathway in An. gambiae cell lines. Interestingly, promoter reporters of these antimicrobial peptides were expressed at very low level in the cell line MSQ43 from Anopheles stephensi. Surprisingly, over-expression of either NF-kappaB transcription factor REL1 or REL2 alone is sufficient to induce the expression of Cec1, Gam1 and Def1. These results suggest that expression of these antimicrobial peptides (AMP) in vivo may be regulated by both the Toll and Imd pathways. We also show here for the first time that Tep4, a gene encoding a thioester containing protein, is regulated by REL2. Taken together, these results suggest that there are significant overlaps of genes regulated by the Toll-Rel1 and Imd-Rel2 pathways. Further, the different expression patterns in two different Anopheline cell lines provide a platform to identify other key positive and negative regulators of the antimicrobial peptide genes.
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