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Involvement of Tiam1, RhoG and ELMO2/ILK in Rac1-mediated phagocytosis in human trabecular meshwork cells.

ABSTRACT: We previously demonstrated that an αvβ5 integrin/FAK- mediated pathway regulated the phagocytic properties of human trabecular meshwork (HTM) cells. Here we demonstrate that this process is mediated by Rac-1 and a previously unreported signaling pathway that utilizes the Tiam1 as well as a novel ILK/RhoG/ELMO2 signaling pathway. Phagocytosis in both a TM-1 cell line and normal HTM cells was mediated by Rac1 and could be significantly decreased by >75% using the Rac1 inhibitor EHop-016. Knockdown of Rac1 in TM-1 cells also inhibited phagocytosis by 40% whereas overexpression of a constitutively active Rac1 or stimulation with PDGF increased phagocytosis by 83% and 32% respectively. Tiam1 was involved in regulating phagocytosis. Knockdown of Tiam1 inhibited phagocytosis by 72% while overexpression of Tiam1 C1199 increased phagocytosis by 75%. Other upstream effectors of Rac1 found to be involved included ELMO2, RhoG, and ILK. Knockdowns of ELMO2, ILK, and RhoG caused a reduction in phagocytosis by 51%, 55% and 46% respectively. In contrast, knockdown of Vav2 and Dock1 or overexpression of Vav2 Y159/172F did not cause a significant change in phagocytosis. These data suggest a novel link between Tiam1 and RhoG/ILK /ELMO2 pathway as upstream effectors of the Rac1-mediated phagocytic process in TM cells.

SUBMITTER: Peotter JL

PROVIDER: S-EPMC5333770 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Involvement of Tiam1, RhoG and ELMO2/ILK in Rac1-mediated phagocytosis in human trabecular meshwork cells.

Peotter Jennifer L JL Phillips Jenny J Tong Tiegang T Dimeo Kaylee K Gonzalez Jose M JM Peters Donna M DM

Experimental cell research 20160815 2

We previously demonstrated that an αvβ5 integrin/FAK- mediated pathway regulated the phagocytic properties of human trabecular meshwork (HTM) cells. Here we demonstrate that this process is mediated by Rac-1 and a previously unreported signaling pathway that utilizes the Tiam1 as well as a novel ILK/RhoG/ELMO2 signaling pathway. Phagocytosis in both a TM-1 cell line and normal HTM cells was mediated by Rac1 and could be significantly decreased by >75% using the Rac1 inhibitor EHop-016. Knockdown ...[more]

PMID: 27539661

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Project description:PurposeContinuous reductions in trabecular meshwork (TM) cellularity inhibit aqueous humor (AH) outflow, which is the main cause of primary open-angle glaucoma. Rho-associated protein kinase inhibitor (ROCKi) targets the TM to reduce intraocular pressure (IOP) and increase AH outflow facility. However, the underlying mechanisms are not entirely clear. Here, we aimed to investigate the effect of a ROCKi (Y-27632) on TM cell proliferation and phagocytosis.MethodsImmortalized human TM (iHTM) cells, glaucomatous TM (GTM3) cells, and primary human TM (pTM) cells were cultured and identified. The effects of various concentrations of Y-27632 on F-actin cytoskeleton were assessed using immunofluorescence. Cell proliferation effects were evaluated using a cell counting kit-8 (CCK8), cell counting, and Ki67 immunostaining. Cell phagocytosis was evaluated using immunofluorescence and flow cytometry in immortalized TM cells. C57BL/6J and Tg-MYOC Y437H mice were used to investigate the proliferative effects of Y-27632 on TM cells in vivo. The effect of Y-27632 on IOP was monitored for 2 weeks, and the outflow facility was detected 2 weeks after IOP measurement. TM cells in mice were counted using immunohistochemistry.ResultsY-27632 (100 μM) significantly promoted the proliferation of both immortal TM cells and pTM cells. In GTM3 cells, phagocytosis was significantly greater in the Y-27632 group than in the control group, nearly reaching the level of phagocytosis in iHTM, as determined using immunofluorescence and flow cytometry. In Tg-MYOC Y437H mice, treatment with Y-27632 significantly decreased IOP and increased outflow facility, which greatly influenced the long-term IOP-lowering effect. The number of TM cells in Tg-MYOC Y437H mice was significantly improved after Y-27632 administration.ConclusionY-27632 promoted cell proliferation and phagocytosis of TM cells, and its proliferative effect was demonstrated in a transgenic mouse model. These results revealed a new IOP-lowering mechanism of Y-27632 through effects on TM cells, suggesting the potential for a correlation between TM cellularity and long-term recovery of IOP.

Project description:PurposeThe purpose of this study is to examine the expression profile of genes related to integrin-mediated phagocytosis that are altered by dexamethasone (DEX) and/or αvβ3 integrin signaling to gain a better understanding of the molecular basis of phagocytosis and the pathophysiology of glucocorticoid-induced ocular hypertension.MethodsRNA and cell lysates were obtained from human trabecular meshwork (HTM) cells incubated with and without DEX for 4-5 d. The relative level of gene expression was evaluated using the Affymetrix Gene Chip® human gene microarray and quantitative PCR (qPCR). Changes in protein expression were validated using western blots or FACS analyses. The involvement of proteins in phagocytosis was determined using siRNA to knock down the expression of these proteins in an immortalized TM-1 cell line. Changes in the phagocytic activity were measured using pHrodo™-labeled S. aureus bioparticles followed by immunofluorescence microscopy. The effect of αvβ3 integrin expression and activity on GULP1 mRNA levels was measured using qPCR in TM-1 cells overexpressing wild type or constitutively active αvβ3 integrin.ResultsGene microarrays revealed statistically significant differences (>2 fold) in the expression of seven genes known to be involved in phagocytosis. Three genes (CD36, ABR, and GULP1) were downregulated, while four genes (ITGB3, CHN1, PIK3R1, and MFGE8) were upregulated. The genes were either associated with modulating RAC1 activity (ABR and CHN1) or integrin signaling (CD36, GULP1, ITGB3, PIK3R1, and MFGE8). Another gene, SIRPA, was also downregulated (1.6 fold) but only in one cell strain. qPCR and western blot analyses verified that DEX caused a decrease in SIRPA and GULP1 mRNA and their protein levels, while levels of CHN1 mRNA and its protein were upregulated by DEX. qPCR showed that although ABR mRNA was downregulated compared to non-treated controls after 5 d of treatment with DEX, no change at the protein level was detected. qPCR analysis also revealed that DEX caused an increase in MFGE8 mRNA levels. The levels of CD36 mRNA and protein varied between cell strains treated with DEX and were not statistically different compared to controls. The knockdown of GULP1 and ABR using siRNAs decreased phagocytosis by 40%. Interestingly, GULP1 mRNA levels were also decreased by 60% when αvβ3 integrin was overexpressed in TM-1 cells.ConclusionThe DEX-induced inhibition of phagocytosis may be caused by the downregulation of ABR and GULP1 disrupting the αvβ5 integrin/RAC1-mediated engulfment pathway. The downregulation of GULP1 by αvβ3 integrin further suggests that this integrin may be a negative regulator of phagocytosis by transcriptionally downregulating proteins needed for phagocytosis. In summary, these results represent new insights into the effects of glucocorticoids and integrin signaling on the phagocytic process in the TM.

Project description:Purpose: Primary open-angle glaucoma (POAG) is the most common glaucoma with elevated intraocular pressure (IOP). Dysfunction of trabecular meshwork (TM) cells affects the outflow facility of aqueous humor, resulting in high IOP. Phagocytosis is an important function for TM cells, this study aims to identify phagocytosis related genes/proteins and the effect of lovastatin in glaucomatous TM cells. Methods: The phagocytic ability of immortal normal human TM cells (iHTM) and glaucomatous TM cells (GTM3) was detected by fluorescent beads and flow cytometry. RNA sequencing and proteomic analysis were used to identify different genes and proteins that potentially involved in regulating the phagocytic function of TM cells. The expression of low density lipoprotein receptor (LDLR) in iHTM and GTM3 was validated by immunofluorescence staining and Western blot. Lovastatin was used to test the effect of LDLR on the phagocytosis of TM cells. The expression of LDLR and the phagocytic ability was measured by western blot and fluorescent beads respectively. Results: The Phagocytosis of GTM3 was significantly weaker than that of iHTM. The integrated analysis upon the transcriptome and proteome identified 3 differentially expressed genes/proteins closely related to phagocytosis of cells, including DAB2: NM_001244871.1, DNAJC6: NM_001256864.1 and LDLR: NM_000527.4. Their expression in GTM3 was obviously lower than that in iHTM. In addition, we have further illustrated the decreased expression of LDLR in GTM3 by immunofluorescence staining and Western blot. However, Lovastatin was found to increase its expression and the phagocytosis ability of GTM3. Conclusions: Three phagocytosis-related genes/proteins were obviously decreased in GTM3 compared to iHTM, they might be responsible for the decreased phagocytic function of TM cells in glaucoma. The up-regulation of LDLR by Lovastatin can enhanced the phagocytosis of GTM3, suggesting Lovastatin might be a new potential medicine to promote the phagocytosis of TM cells in glaucoma.

Dataset Information

Involvement of Tiam1, RhoG and ELMO2/ILK in Rac1-mediated phagocytosis in human trabecular meshwork cells.

Publications

Involvement of Tiam1, RhoG and ELMO2/ILK in Rac1-mediated phagocytosis in human trabecular meshwork cells.

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