Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis.

Purpose: The 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.

Methods: RNA 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 bioparticles followed by immunofluorescence microscopy. The effect of αvβ3 integrin expression and activity on 1 mRNA levels was measured using qPCR in TM-1 cells overexpressing wild type or constitutively active αvβ3 integrin.

Results: Gene microarrays revealed statistically significant differences (>2 fold) in the expression of seven genes known to be involved in phagocytosis. Three genes (, , and ) were downregulated, while four genes (, , , and ) were upregulated. The genes were either associated with modulating RAC1 activity ( and ) or integrin signaling (, , , , and ). Another gene, , was also downregulated (1.6 fold) but only in one cell strain. qPCR and western blot analyses verified that DEX caused a decrease in and mRNA and their protein levels, while levels of mRNA and its protein were upregulated by DEX. qPCR showed that although 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 mRNA levels. The levels of mRNA and protein varied between cell strains treated with DEX and were not statistically different compared to controls. The knockdown of and using siRNAs decreased phagocytosis by 40%. Interestingly, mRNA levels were also decreased by 60% when αvβ3 integrin was overexpressed in TM-1 cells.

Conclusion: The 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.