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Glucocorticoids with different chemical structures but similar glucocorticoid receptor potency regulate subsets of common and unique genes in human trabecular meshwork cells.

Nehmé A, Lobenhofer EK, Stamer WD, Edelman JL - BMC Med Genomics (2009)

Bottom Line: Using Ingenuity Pathway Analysis (IPA) software, analysis of the data from TM 86 cells showed that DEX significantly regulated transcripts associated with RNA post-transcriptional modifications, whereas FA and TA modulated genes involved in lipid metabolism and cell morphology, respectively.In TM 93 cells, DEX significantly regulated genes implicated in histone methylation, whereas FA and TA altered genes associated with cell cycle and cell adhesion, respectively.These GC structure-dependent effects appear to be TM cell-lineage dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Allergan, Inc,, Irvine, CA 92612, USA. nehme_alissar@allergan.com

ABSTRACT

Background: In addition to their well-documented ocular therapeutic effects, glucocorticoids (GCs) can cause sight-threatening side-effects including ocular hypertension presumably via morphological and biochemical changes in trabecular meshwork (TM) cells. In the present study, we directly compared the glucocorticoid receptor (GR) potency for dexamethasone (DEX), fluocinolone acetonide (FA) and triamcinolone acetonide (TA), examined the expression of known GRalpha and GRbeta isoforms, and used gene expression microarrays to compare the effects of DEX, FA, and TA on the complete transcriptome in two primary human TM cell lines.

Methods: GR binding affinity for DEX, FA, and TA was measured by a cell-free competitive radio-labeled GR binding assay. GR-mediated transcriptional activity was assessed using the GeneBLAzer beta-lactamase reporter gene assay. Levels of GRalpha and GRbeta isoforms were assessed by Western blot. Total RNA was extracted from TM 86 and TM 93 cells treated with 1 muM DEX, FA, or TA for 24 hr and used for microarray gene expression analysis. The microarray experiments were repeated three times. Differentially expressed genes were identified by Rosetta Resolver Gene Expression Analysis System.

Results: The GR binding affinity (IC50) for DEX, FA, and TA was 5.4, 2.0, and 1.5 nM, respectively. These values are similar to the GR transactivation EC50 of 3.0, 0.7, and 1.5 nM for DEX, FA, and TA, respectively. All four GRalpha translational isoforms (A-D) were expressed in TM 86 and TM 93 total cell lysates, however, the C and D isoforms were more highly expressed relative to A and B. All four GRbeta isoforms (A-D) were also detected in TM cells, although GRbeta-D isoform expression was lower compared to that of the A, B, or C isoforms. Microarray analysis revealed 1,968 and 1,150 genes commonly regulated by DEX, FA, and TA in TM 86 and TM 93, respectively. These genes included RGC32, OCA2, ANGPTL7, MYOC, FKBP5, SAA1 and ZBTB16. In addition, each GC specifically regulated a unique set of genes in both TM cell lines. Using Ingenuity Pathway Analysis (IPA) software, analysis of the data from TM 86 cells showed that DEX significantly regulated transcripts associated with RNA post-transcriptional modifications, whereas FA and TA modulated genes involved in lipid metabolism and cell morphology, respectively. In TM 93 cells, DEX significantly regulated genes implicated in histone methylation, whereas FA and TA altered genes associated with cell cycle and cell adhesion, respectively.

Conclusion: Human trabecular meshwork cells in culture express all known GRalpha and GRbeta translational isoforms, and GCs with similar potency but subtly different chemical structure are capable of regulating common and unique gene subsets and presumably biologic responses in these cells. These GC structure-dependent effects appear to be TM cell-lineage dependent.

No MeSH data available.


Related in: MedlinePlus

The most prominently affected gene network in response to DEX in TM 86 cells. Network was classified as: RNA-Post-Transcriptional Modification, Antigen Presentation, Immune cell Trafficking. Regulation of this network in TM 86 cells is compared across treatment with DEX, FA or TA. Pathway contains RNA binding protein S1 (RNPS1), which occupies a central position in this network. Red: induction; green: repression; white: unaffected; color intensity correlates with fold change; number: fold change. The diagram was obtained from Ingenuity Pathway Analysis (Ingenuity® Systems).
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Figure 5: The most prominently affected gene network in response to DEX in TM 86 cells. Network was classified as: RNA-Post-Transcriptional Modification, Antigen Presentation, Immune cell Trafficking. Regulation of this network in TM 86 cells is compared across treatment with DEX, FA or TA. Pathway contains RNA binding protein S1 (RNPS1), which occupies a central position in this network. Red: induction; green: repression; white: unaffected; color intensity correlates with fold change; number: fold change. The diagram was obtained from Ingenuity Pathway Analysis (Ingenuity® Systems).

Mentions: Using Ingenuity Pathway Analysis, we identified networks that were significantly enriched using the lists of differentially expressed genes. This analysis confirmed the unique regulation of a functional subset of genes by each of the GCs in TM 86 and TM 93 cells. For example, in TM 86, DEX significantly regulated a network centered around the RNA binding protein S1 or RNPS1 (Figure 5), whereas FA significantly modulated a GR-dependent network (data not shown). In TM 93, DEX significantly affected a network centered around a histone methyltransferase known as suppressor of zeste 12 homolog or SUZ12 (data not shown), whereas FA regulated a transcriptional repressor referred to as jumonji, AT rich interactive domain 1B or JARID1B-based network (Figure 6).


Glucocorticoids with different chemical structures but similar glucocorticoid receptor potency regulate subsets of common and unique genes in human trabecular meshwork cells.

Nehmé A, Lobenhofer EK, Stamer WD, Edelman JL - BMC Med Genomics (2009)

The most prominently affected gene network in response to DEX in TM 86 cells. Network was classified as: RNA-Post-Transcriptional Modification, Antigen Presentation, Immune cell Trafficking. Regulation of this network in TM 86 cells is compared across treatment with DEX, FA or TA. Pathway contains RNA binding protein S1 (RNPS1), which occupies a central position in this network. Red: induction; green: repression; white: unaffected; color intensity correlates with fold change; number: fold change. The diagram was obtained from Ingenuity Pathway Analysis (Ingenuity® Systems).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749862&req=5

Figure 5: The most prominently affected gene network in response to DEX in TM 86 cells. Network was classified as: RNA-Post-Transcriptional Modification, Antigen Presentation, Immune cell Trafficking. Regulation of this network in TM 86 cells is compared across treatment with DEX, FA or TA. Pathway contains RNA binding protein S1 (RNPS1), which occupies a central position in this network. Red: induction; green: repression; white: unaffected; color intensity correlates with fold change; number: fold change. The diagram was obtained from Ingenuity Pathway Analysis (Ingenuity® Systems).
Mentions: Using Ingenuity Pathway Analysis, we identified networks that were significantly enriched using the lists of differentially expressed genes. This analysis confirmed the unique regulation of a functional subset of genes by each of the GCs in TM 86 and TM 93 cells. For example, in TM 86, DEX significantly regulated a network centered around the RNA binding protein S1 or RNPS1 (Figure 5), whereas FA significantly modulated a GR-dependent network (data not shown). In TM 93, DEX significantly affected a network centered around a histone methyltransferase known as suppressor of zeste 12 homolog or SUZ12 (data not shown), whereas FA regulated a transcriptional repressor referred to as jumonji, AT rich interactive domain 1B or JARID1B-based network (Figure 6).

Bottom Line: Using Ingenuity Pathway Analysis (IPA) software, analysis of the data from TM 86 cells showed that DEX significantly regulated transcripts associated with RNA post-transcriptional modifications, whereas FA and TA modulated genes involved in lipid metabolism and cell morphology, respectively.In TM 93 cells, DEX significantly regulated genes implicated in histone methylation, whereas FA and TA altered genes associated with cell cycle and cell adhesion, respectively.These GC structure-dependent effects appear to be TM cell-lineage dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Allergan, Inc,, Irvine, CA 92612, USA. nehme_alissar@allergan.com

ABSTRACT

Background: In addition to their well-documented ocular therapeutic effects, glucocorticoids (GCs) can cause sight-threatening side-effects including ocular hypertension presumably via morphological and biochemical changes in trabecular meshwork (TM) cells. In the present study, we directly compared the glucocorticoid receptor (GR) potency for dexamethasone (DEX), fluocinolone acetonide (FA) and triamcinolone acetonide (TA), examined the expression of known GRalpha and GRbeta isoforms, and used gene expression microarrays to compare the effects of DEX, FA, and TA on the complete transcriptome in two primary human TM cell lines.

Methods: GR binding affinity for DEX, FA, and TA was measured by a cell-free competitive radio-labeled GR binding assay. GR-mediated transcriptional activity was assessed using the GeneBLAzer beta-lactamase reporter gene assay. Levels of GRalpha and GRbeta isoforms were assessed by Western blot. Total RNA was extracted from TM 86 and TM 93 cells treated with 1 muM DEX, FA, or TA for 24 hr and used for microarray gene expression analysis. The microarray experiments were repeated three times. Differentially expressed genes were identified by Rosetta Resolver Gene Expression Analysis System.

Results: The GR binding affinity (IC50) for DEX, FA, and TA was 5.4, 2.0, and 1.5 nM, respectively. These values are similar to the GR transactivation EC50 of 3.0, 0.7, and 1.5 nM for DEX, FA, and TA, respectively. All four GRalpha translational isoforms (A-D) were expressed in TM 86 and TM 93 total cell lysates, however, the C and D isoforms were more highly expressed relative to A and B. All four GRbeta isoforms (A-D) were also detected in TM cells, although GRbeta-D isoform expression was lower compared to that of the A, B, or C isoforms. Microarray analysis revealed 1,968 and 1,150 genes commonly regulated by DEX, FA, and TA in TM 86 and TM 93, respectively. These genes included RGC32, OCA2, ANGPTL7, MYOC, FKBP5, SAA1 and ZBTB16. In addition, each GC specifically regulated a unique set of genes in both TM cell lines. Using Ingenuity Pathway Analysis (IPA) software, analysis of the data from TM 86 cells showed that DEX significantly regulated transcripts associated with RNA post-transcriptional modifications, whereas FA and TA modulated genes involved in lipid metabolism and cell morphology, respectively. In TM 93 cells, DEX significantly regulated genes implicated in histone methylation, whereas FA and TA altered genes associated with cell cycle and cell adhesion, respectively.

Conclusion: Human trabecular meshwork cells in culture express all known GRalpha and GRbeta translational isoforms, and GCs with similar potency but subtly different chemical structure are capable of regulating common and unique gene subsets and presumably biologic responses in these cells. These GC structure-dependent effects appear to be TM cell-lineage dependent.

No MeSH data available.


Related in: MedlinePlus