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 glucocort...
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| Format: | Article |
| Language: | English |
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Molecular Vision
2019-04-01
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| Series: | Molecular Vision |
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| Online Access: | http://www.molvis.org/molvis/v25/237/ |
| _version_ | 1818806688803717120 |
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| author | Jennifer A. Faralli Harini Desikan Jennifer Peotter Nitin Kanneganti Benjamin Weinhaus Mark S. Filla Donna M. Peters |
| author_facet | Jennifer A. Faralli Harini Desikan Jennifer Peotter Nitin Kanneganti Benjamin Weinhaus Mark S. Filla Donna M. Peters |
| author_sort | Jennifer A. Faralli |
| collection | DOAJ |
| description | 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 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.
Results: Gene 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.
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. |
| first_indexed | 2024-12-18T19:13:45Z |
| format | Article |
| id | doaj.art-fdcc00f626fc4c24860bf77abc29ff89 |
| institution | Directory Open Access Journal |
| issn | 1090-0535 1090-0535 |
| language | English |
| last_indexed | 2024-12-18T19:13:45Z |
| publishDate | 2019-04-01 |
| publisher | Molecular Vision |
| record_format | Article |
| series | Molecular Vision |
| spelling | doaj.art-fdcc00f626fc4c24860bf77abc29ff892022-12-21T20:56:10ZengMolecular VisionMolecular Vision1090-05351090-05352019-04-01251237254Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosisJennifer A. Faralli0Harini Desikan1Jennifer Peotter2Nitin Kanneganti3Benjamin Weinhaus4Mark S. Filla5Donna M. Peters6Department of Pathology & Laboratory Medicine, University of Wisconsin, Madison, WIDepartment of Pathology & Laboratory Medicine, University of Wisconsin, Madison, WIDepartment of Pathology & Laboratory Medicine, University of Wisconsin, Madison, WIDepartment of Pathology & Laboratory Medicine, University of Wisconsin, Madison, WIDepartment of Pathology & Laboratory Medicine, University of Wisconsin, Madison, WIDepartment of Pathology & Laboratory Medicine, University of Wisconsin, Madison, WIDepartment of Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WIPurpose: 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 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. Results: Gene 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. 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.http://www.molvis.org/molvis/v25/237/human trabecular meshworkdexamethasonephagocytosis |
| spellingShingle | Jennifer A. Faralli Harini Desikan Jennifer Peotter Nitin Kanneganti Benjamin Weinhaus Mark S. Filla Donna M. Peters Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis Molecular Vision human trabecular meshwork dexamethasone phagocytosis |
| title | Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis |
| title_full | Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis |
| title_fullStr | Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis |
| title_full_unstemmed | Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis |
| title_short | Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis |
| title_sort | genomic proteomic analyses of dexamethasone treated human trabecular meshwork cells reveal a role for gulp1 and abr in phagocytosis |
| topic | human trabecular meshwork dexamethasone phagocytosis |
| url | http://www.molvis.org/molvis/v25/237/ |
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