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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.


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Western blot analysis of the GRα (A) and GRβ (B) isoforms in human TM86 and TM93. Blots resolving 45 μg of total cell lysates from HeLa, TM 86, or TM 93 cells detected with anti-GRα antibody (PA1-516) or anti-GRβ antibody (PA3-514) are shown. Three independent cell lysates were analyzed per cell line. Additional bands in between GR-C and GR-D isoforms may be degradation products or cell-specific isoforms.
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Figure 1: Western blot analysis of the GRα (A) and GRβ (B) isoforms in human TM86 and TM93. Blots resolving 45 μg of total cell lysates from HeLa, TM 86, or TM 93 cells detected with anti-GRα antibody (PA1-516) or anti-GRβ antibody (PA3-514) are shown. Three independent cell lysates were analyzed per cell line. Additional bands in between GR-C and GR-D isoforms may be degradation products or cell-specific isoforms.

Mentions: The GR transactivation potency for DEX, FA and TA has never been directly compared in one cell type. Moreover, a wide range of GR transactivation potency values have been reported in the literature for each of these three GCs [35]. Therefore, GR transactivation potency was assessed in the GeneBLAzer β-lactamase reporter gene technology in MMTV-bla HeLa CellSensor® cells (Invitrogen). Attempts to establish a robust and reproducible GeneBLAzer assay using primary human TM cells failed, and HeLa cells seemed a reasonable surrogate since the levels of all GRα and GRβ isoforms in these cells were comparable to those in TM 86 and TM 93 cells (Figure 1). HeLa cells were treated with various concentrations of GC for 5 hr. Figure 2 shows that all three GCs induced an increase in GR transactivation activity in a dose-dependent manner. The EC50 (concentration causing a half-maximal increase in GR transactivation) values for DEX, FA, and TA, were 3.02 ± 0.02, 0.67 ± 0.10, and 1.29 ± 0.28 nM, respectively. These data suggest that all three GCs are roughly equivalent at stimulating GR transcriptional activity. The expression of multiple GRα isoforms in HeLa cells has been previously reported [20] and confirmed in our study. Assuming that different GRα isoforms might exhibit different transcriptional activities in response to each GC, the observed GR-mediated transactivation function is the aggregate response generated by multiple GR isoforms in these cells.


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)

Western blot analysis of the GRα (A) and GRβ (B) isoforms in human TM86 and TM93. Blots resolving 45 μg of total cell lysates from HeLa, TM 86, or TM 93 cells detected with anti-GRα antibody (PA1-516) or anti-GRβ antibody (PA3-514) are shown. Three independent cell lysates were analyzed per cell line. Additional bands in between GR-C and GR-D isoforms may be degradation products or cell-specific isoforms.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Western blot analysis of the GRα (A) and GRβ (B) isoforms in human TM86 and TM93. Blots resolving 45 μg of total cell lysates from HeLa, TM 86, or TM 93 cells detected with anti-GRα antibody (PA1-516) or anti-GRβ antibody (PA3-514) are shown. Three independent cell lysates were analyzed per cell line. Additional bands in between GR-C and GR-D isoforms may be degradation products or cell-specific isoforms.
Mentions: The GR transactivation potency for DEX, FA and TA has never been directly compared in one cell type. Moreover, a wide range of GR transactivation potency values have been reported in the literature for each of these three GCs [35]. Therefore, GR transactivation potency was assessed in the GeneBLAzer β-lactamase reporter gene technology in MMTV-bla HeLa CellSensor® cells (Invitrogen). Attempts to establish a robust and reproducible GeneBLAzer assay using primary human TM cells failed, and HeLa cells seemed a reasonable surrogate since the levels of all GRα and GRβ isoforms in these cells were comparable to those in TM 86 and TM 93 cells (Figure 1). HeLa cells were treated with various concentrations of GC for 5 hr. Figure 2 shows that all three GCs induced an increase in GR transactivation activity in a dose-dependent manner. The EC50 (concentration causing a half-maximal increase in GR transactivation) values for DEX, FA, and TA, were 3.02 ± 0.02, 0.67 ± 0.10, and 1.29 ± 0.28 nM, respectively. These data suggest that all three GCs are roughly equivalent at stimulating GR transcriptional activity. The expression of multiple GRα isoforms in HeLa cells has been previously reported [20] and confirmed in our study. Assuming that different GRα isoforms might exhibit different transcriptional activities in response to each GC, the observed GR-mediated transactivation function is the aggregate response generated by multiple GR isoforms in these cells.

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