Regulatory Effects of GPR158 Overexpression in Trabecular Meshwork Cells of the Eye’s Aqueous Outflow Pathways
Abstract
:1. Introduction
2. Results
2.1. Profiling of GPR158-Regulated Gene Expression
2.2. IPA Upstream Stress Regulator Analysis
2.2.1. Upstream Stress Regulators: Dexamethasone and TGFB1 (Fibrosis)
2.2.2. Upstream Stress Regulators: XBP1 and ATF4 (UPR)
2.2.3. Upstream Stress Regulator: TP53
2.3. Functional Studies
2.4. Upstream GPCR Regulator ADRB1 and Other Genes Linked to GPCR Signaling
3. Discussion
4. Materials and Methods
4.1. Antibodies
4.2. Immortalized Trabecular Meshwork Cell Line and Lentivirus-Transformed GPR158 Overexpression Model
4.3. Human Trabecular Meshwork Primary Cell Strains and Transient Transfection
4.4. Microarray-Based Gene Expression Profiling
4.5. Ingenuity Pathway Analysis
4.6. Quantitative Polymerase Chain Reaction (qPCR)
4.7. Immunoblotting
4.8. Calcification Assay
4.9. Mouse Model of Steroid-Induced Ocular Hypertension
4.10. Statistical Analysis and Reproducibility
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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GPR158 Dose | Exp 1 | Exp 2A | Exp 2B |
---|---|---|---|
Doxycycline | Low | High | High |
Duration | 72 h | 24 h | 96 h |
IPA Top Upstream Regulators | |||
Dexamethasone | p = 2.71 × 10−9 | p = 4.55 × 10−17 | p = 1.62 × 10−6 |
activated | |||
TGFB1 | p = 1.19 × 10−9 | p = 1.02 × 10−23 | p = 2.54 × 10−16 |
inhibited | |||
XBP1 | p = 1.09 × 10−5 | ||
inhibited | |||
ATF4 | p = 5.46 × 10−6 | ||
TP53 | p = 1.13 × 10−5 | p = 4.84 × 10−18 | p = 3.41 × 10−15 |
inhibited | activated |
Gene | Fold Change in Gene Expression | Change Direction Predicted | Change Direction Observed | ||
---|---|---|---|---|---|
Exp 1 Low, 72 h | Exp 2A High, 24 h | Exp 2B High, 96 h | |||
Dexamethasone regulated | |||||
Acute | |||||
AKR1C1/C2 | −1.302 | −1.991 | −1.503 | up | down |
AKR1C3 | −1.443 | −1.625 | up | down | |
AKR1C4 * | −1.313 | −2.206 | −1.576 | up * | down |
CA12 ** | −1.317 | −1.727 | −1.362 | up | down |
CHI3L1 | −1.370 | −1.571 | up | down | |
Extracellular matrix | |||||
ADAMTS1 | −1.229 | up | down | ||
COL1A1 | −1.250 | up | down | ||
COL4A1 | −1.276 | −1.256 | up | down | |
COL4A5 | +1.256 | up | up | ||
COL10A1 | +1.686 | +3.183 | down | up | |
LAMA4 | −1.218 | −1.218 | −1.212 | up | down |
LAMA5 | −1.212 | −1.394 | +1.220 | up | regulates |
VCAN | −1.235 | −1.570 | −1.786 | down | down |
Growth factors and matricellular proteins | |||||
CTGF | −1.144 | −1.320 | up | down | |
CYR61 | −1.211 | −1.292 | +1.315 | regulates | regulates |
GDF15 | +1.354 | +1.374 | down | up | |
TGFB2 | −1.443 | up | down | ||
THBS1 | −1.242 | −1.363 | +1.249 | up | regulates |
WNT5A | −1.264 | up | down | ||
WNT5B *** | −1.212 | −1.376 | --- | down | |
TGFB1 regulated | |||||
Extracellular matrix | |||||
COL1A1 | −1.250 | up | down | ||
COL3A1 | −1.594 | up | down | ||
COL4A1 | −1.276 | −1.256 | up | down | |
COL5A1 | −1.254 | up | down | ||
COL6A3 | −1.539 | up | down | ||
VCAN | −1.570 | −1.786 | down | down | |
Growth factors and matricellular proteins | |||||
BMP4 | −1.212 | −1.382 | down | down | |
DACT3 **** | +1.457 | +1.228 | --- | up | |
DKK3 | +1.262 | up | up | ||
GDF15 | +1.354 | +1.374 | up | up | |
THBS1 | −1.242 | −1.363 | +1.249 | up | regulates |
Gene | Fold Change in Gene Expression | Change Direction Predicted | Change Direction Observed | ||
---|---|---|---|---|---|
Exp 1 Low, 72 h | Exp 2A High, 24 h | Exp 2B High, 96 h | |||
Calcification related | |||||
ACTG2 | −1.204 | −1.414 | up | down | |
ALPL | +1.340 | up | up | ||
ALPP | +1.578 | up | up | ||
BGLAP | --- | --- | |||
BMP2 | up | --- | |||
BMP4 | −1.212 | −1.382 | up | down | |
COL1A1 | −1.250 | up | down | ||
COL10A1 | +1.686 | +3.183 | up | up | |
ECM1 | +1.319 | up | up | ||
MGP | −1.221 | down | down | ||
OSTF1 | +1.203 | down | up | ||
SLC8A2 | +2.174 | +1.777 | down | up | |
TNFRSF11B | −1.215 | down | down | ||
WNT5A | −1.264 | up | down | ||
WNT5B | −1.212 | −1.376 | up | down |
Gene | Fold Change in Gene Expression | Change Direction Predicted | Change Direction Observed | ||
---|---|---|---|---|---|
Exp 1 Low, 72 h | Exp 2A High, 24 h | Exp 2B High, 96 h | |||
TP53-regulated | |||||
Cell cycle progression | |||||
CCNA1 ***** | −1.956 | --- | down | ||
CCNA2 | −1.368 | down | down | ||
CCNB2 | −1.269 | regulates | down | ||
CCND1 | −1.292 | regulates | down | ||
CCNE1 | −1.258 | regulates | down | ||
CDC25C | −1.371 | down | down | ||
MYC | −1.707 | −1.268 | down | down | |
Cell cycle arrest | |||||
BRSK1 | +1.475 | +1.335 | up | up | |
CDC42EP5 | +1.708 | +1.547 | up | up | |
CDKN1A | −1.186 | +1.264 | up | up | |
GAS1 | −1.516 | −1.513 | regulates | down | |
KLF6 | −1.818 | up | down | ||
MAPRE3 | −1.357 | +1.219 | up | regulates | |
SIRT4 | +1.433 | +1.306 | up | up | |
TP53 | up | --- | |||
TXNIP | −1.427 | +9.300 | up | regulates | |
Mitosis | |||||
CDC20 | −1.255 | down | down | ||
CENPF | −2.203 | down | down | ||
CEP55 | −1.236 | down | down | ||
AURKA | −1.259 | down | down | ||
PEG10 | −1.235 | −1.410 | down | down |
Gene | Fold Change in Gene Expression | Function | ||
---|---|---|---|---|
Exp 1 Low, 72 h | Exp 2A High, 24 h | Exp 2B High, 96 h | ||
GPCR signaling | ||||
GNB5 | +1.281 | G protein | ||
GNG7 | +1.360 | +1.26 | G protein | |
RGS4 | −2.778 | −2.21 | GTPase activating | |
PLCG1 | −1.387 | Phospholipase C | ||
PLCH2 | +1.265 | Phospholipase C |
Antigen | Where Purchased | Catalogue Number | Species |
---|---|---|---|
GPR158 | Sigma-Aldrich Corporation, St Louis, MO, USA | Cat# HPA013185 | Rabbit |
MYOC | Santa Cruz Biotechnology, Santa Cruz, CA, USA | Cat# sc-137233 | Mouse |
TP53 | Cell Signaling Technology, Danvers, MA, USA | Cat# IC12 | Mouse |
COL10A1 | Invitrogen, Waltham, MA, USA | Cat# PA5-49198 | Rabbit |
SLCA8 | Origene Technologies, Rockville, MD, USA | Cat# TA328916 | Rabbit |
HSPA5 (GRP78/BiP) | Abcam, Cambridge, UK | Cat# ab212054 | Mouse |
ATF4 | Cell Signaling Technology, Danvers, MA, USA | Cat# 11815 | Rabbit |
DDIT3 (CHOP) | Cell Signaling Technology, Danvers, MA, USA | Cat# 5554 | Rabbit |
ACTB | Abcam, Cambridge, UK | Cat# ab6276 | Mouse |
GAPDH | Cell Signaling Technology, Danvers, MA, USA | Cat# 97166 | Mouse |
Gene | Primer Sequence |
---|---|
TGFB2 | Forward: GCGACGAAGAGTACTACGCC Reverse: TGGCATCAAGGTACCCACAG |
COL10A1 | Forward: CAAGGCACCATCTCCAGG AA Reverse: AAAGGGTAT TTGTGGCAGCATATT |
CA12 | Forward: ACTGCGGCAGGACTGAGTCT Reverse: CACAATACAGATGCCAAGAATGC |
TP53 | Forward: TTTTCCCCTCCCATGTGCTC Reverse: TGGACGGTGGCTCTAGACTT |
FN1 | Forward: AGCGGACCTACCTAGGCAAT Reverse: GGTTTGCGATGGTACAGCTT |
HSPA5 | Forward: CCAAGAGAGGGTTCTTGAATCTCG Reverse: ATGGGCCAGCCTGGATATACAACA |
sXBP1 | Forward: CTGAGTCCGAATCAGGTGCAG Reverse: ATCCATGGGGAGATGTTCTGG |
ATF4 | Forward: CAGCACAGCCCCTCTACCA Reverse: GCCCGCCTTAGCCTTGTC |
DDIT3 | Forward: AGAACCAGGAAACGGAAACAGA Reverse: TCTCCTTCATGCGCTGCTTT |
ACTB | Forward: GTCATTCCAAATATGAGATGCGT Reverse: GCTATCACCTCCCCTGTGTG |
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Suarez, M.F.; Itakura, T.; Pany, S.; Jeong, S.; Chintala, S.K.; Raizman, M.B.; Riesinger, S.; Lazarova, T.; Echenique, J.; Serra, H.M.; et al. Regulatory Effects of GPR158 Overexpression in Trabecular Meshwork Cells of the Eye’s Aqueous Outflow Pathways. Stresses 2023, 3, 629-652. https://doi.org/10.3390/stresses3030044
Suarez MF, Itakura T, Pany S, Jeong S, Chintala SK, Raizman MB, Riesinger S, Lazarova T, Echenique J, Serra HM, et al. Regulatory Effects of GPR158 Overexpression in Trabecular Meshwork Cells of the Eye’s Aqueous Outflow Pathways. Stresses. 2023; 3(3):629-652. https://doi.org/10.3390/stresses3030044
Chicago/Turabian StyleSuarez, Maria Fernanda, Tatsuo Itakura, Satyabrata Pany, Shinwu Jeong, Shravan K. Chintala, Michael B. Raizman, Steven Riesinger, Tsvetelina Lazarova, José Echenique, Horacio M. Serra, and et al. 2023. "Regulatory Effects of GPR158 Overexpression in Trabecular Meshwork Cells of the Eye’s Aqueous Outflow Pathways" Stresses 3, no. 3: 629-652. https://doi.org/10.3390/stresses3030044