Abstract
Purpose :
GPR158, a distant member of the G protein coupled receptor family C, was identified by our lab in a genomic screen for genes associated with susceptibility to glucocorticoid (GC)-induced ocular hypertension (OH). GPR158 is expressed in the trabecular meshwork (TM) and in TM cell cultures, and expression is stimulated by GC treatment (PMID: 23451275). In this study, we investigated the effects of GPR158 expression levels in trabecular meshwork cells.
Methods :
An immortalized TM cell line (TM1) stably-transduced with GPR158 under doxycycline (Dox) regulation was created. For GPR158 over-expression, cells were treated with low Dox (100 ng/mL) or high Dox (500 ng/mL) for up to 96hs. For GPR158 knockdown, TM1 cells were transfected with a pool of three custom designed siRNA oligonucleotides. Gene expression microarray profiling was performed in over-expressing cells, and evaluated by Ingenuity Pathway Analysis (IPA). A group of regulated genes was further evaluated by qPCR and western blot blotting in both over-expressing and knockdown cells. Statistical significance was calculated using Student’s t-test (N=3; P≤0.05 considered significant).
Results :
IPA revealed that GPR158 over-expression downregulates genes related to ECM deposition. Validation by qPCR showed that with low Dox, expression of ECM-associated genes TGFB2, COL1A1 and FN is downregulated (p>0.05), while SMAD3 is upregulated (p>0.05). At high Dox, TGFB2 and COL1A1 are still downregulated (p= 0.0109 and 0.002 respectively), but expression of FN and SMAD3 is increased (p>0.05 and p=0.01 respectively). Somewhat different behaviors were observed at the protein level. At low Dox, TGFB2 protein is decreased, but with high Dox it is increased (p>0.05). FN protein is upregulated at all conditions (p=0.028). SMAD3 protein is only increased with high Dox, but activated phosphorylated p-SMAD3 is decreased (p>0.05, both). When GPR158 expression was knocked down, TGFB2 was downregulated (~55%, p=0.037), but COL1A1, FN and SMAD3 did not show any changes
Conclusions :
GPR158 modulates expression of genes associated with extracellular matrix deposition. This suggests a possible mechanism by which changes in GPR158 expression levels could contribute to SIOH susceptibility.
This is a 2020 ARVO Annual Meeting abstract.