Journal: bioRxiv
Article Title: Nocturnal intraocular pressure rise is regulated by norepinephrine via RHOB
doi: 10.1101/2025.03.21.644679
Figure Lengend Snippet: A RHOB knockout (KO) was achieved by transfection of CRISPR/Cas9 plasmid. B Western blot analysis of WT RHOB and RHOB-KO in iiHTMCs. C Cell growth of RHOB-KO iHTMC. KO had no effect on cell growth ( p > 0.05). D Effect of dobutamine on RHOB KO cell viability. Incubation with dobutamine for 6 h dose-dependently decreased cell viability (two-way ANOVA [concentration; * p = 0.0190]), whereas RHOB KO showed no effect. E Effect of 2 d of dobutamine treatment (0–10 μM) on TM aging. Low concentrations of dobutamine (10–100 nM) increased iHTMC senescence (* p < 0.05, *** p < 0.001, vs. DMSO, Dunnett’s multiple comparison test, two-way ANOVA [interaction, * p = 0.0122; time, ** p = 0.0073; coating, p < 0.2293]). F Effect of dobutamine on TM phagocytosis in RHOB KO cells. RHOB KO rescued dobutamine-suppressed TM phagocytosis (* p < 0.05, vs. WT, Šidák multiple comparison test, two-way ANOVA [concentration; * p = 0.0378, KO; ** p < 0.0012]). G Dobutamine gradually increased TM F-actin levels, but RHOB KO showed no effect (**** p < 0.0001 vs. DMSO, Dunnett’s multiple comparison test, two-way ANOVA [concentration, ** p = 0.0027; KO, p = 0.9508]). H Effect of dobutamine on TM aging in RHOB-KO cells. KO showed slightly decreased TM senescence at high dobutamine levels (** p < 0.01 vs. DMSO, Dunnett’s multiple comparison test, two-way ANOVA [interaction, * p = 0.0122; concentration; ** p = 0.0073; KO, p = 0.2293]). Data were normalized using signals from DMSO-treated cells and presented as bar graphs (mean ± SEM) with scatter plot of independent experiments (n = 3–4). I ROCK inhibitor Y27632 and RHO inhibitor rhosin rescued dobutamine-attenuated TM phagocytosis (* p < 0.05, vs. WT, Šidák multiple comparison test, two-way ANOVA [interaction, * p = 0.0342; concentration, ** p = 0.0030; KO, ** p = 0.0029]). J Lysophosphatidic acid (LPA), a RHO activator, dose-dependently attenuated TM phagocytosis (* p < 0.05 vs. DMSO, Dunnett’s multiple comparison test, one-way ANOVA [* p = 0.0202]). K Constitutively active (CA) RHOB (G14V in the GDP/GTP binding site) and RHOC WT structures. L Overexpression of RHOB WT and CA significantly inhibited phagocytosis, which was rescued by rhosin (* p < 0.05, vs. DMSO, Dunnett’s multiple comparison test, one-way ANOVA [* p = 0.0413]). Overexpression of WT RHOC had no effect on phagocytosis. N RHOB overexpression dramatically increased the number of adherent cells (*** p < 0.001 vs. DMSO, Dunnett’s multiple comparison test, one-way ANOVA [** p = 0.0002]). O RHOB overexpression inhibits liquid permeability in iHTMC multilayers (** p < 0.01, unpaired t-test). P Dobutamine-attenuated TM permeability was not observed in RHOB KO iHTMC multilayers (* p < 0.05, unpaired t -test).
Article Snippet: After destaining, membranes were incubated with rabbit polyclonal antibody against RHOB (1:1,000; 14326-1-AP, ProteinTech).
Techniques: Knock-Out, Transfection, CRISPR, Plasmid Preparation, Western Blot, Incubation, Concentration Assay, Comparison, Binding Assay, Over Expression, Permeability
Proteintech is a verified supplier 
![A,B Top 10 signatures from kinase perturbations extracted from GEO down and GEO up, respectively, linked by the combined score with p-values and gene counts. C Pathway enrichment analysis of the Reactome using 18 genes revealed several RHO-related indices D <t>RHOB</t> GTPase activity assay of NE-treated iHTMCs showed that NE enhanced RHOB activity (unpaired t -test, * p < 0.05). Data are presented as bar graphs (mean ± SEM) with scatter plots of independent experiments (n = 4). E RhoB protein expression in mouse eyes exposed to L-NE for 5 h. Coomassie Brilliant Blue staining was performed to confirm total protein levels. F RHOB induction in iHTMC 3, 6, and 9 h after L-NE administration. G Relative RHOB levels by NE exposure time. RHOB expression gradually increased with NE exposure, peaking at 6 h after stimulation (* p < 0.05 vs. WT, Šidák multiple comparison test, two-way ANOVA [interaction, * p = 0.0419; time, p = 0.0597; DMSO vs. NE, *** p = 0.0007]).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_79/10__1101_slash_2025__03__21__644679/10__1101_slash_2025__03__21__644679___F2.large.jpg)
![A Transcriptional factor (TF) enrichment analysis of ENCODE and ChEA Consensus TFs from ChIP-X. The cyclic adenosine monophosphate (cAMP)-responsive element (CRE)- binding protein CREB1 has been identified as the most important TF. B Upset plot of the cAMP-related five gene sets obtained from GSE21727, GSE12056, 25313160, GSE5041, and PRJDB20330. Only RHOB was identified as the common variable gene. Black points indicate intersections and gray points indicate no intersections. C Annotation analysis of the human RHOB promoter (−1,500 to +1 bp) using the UCSC genome browser. The TATA box, CAAT element, and a previously unknown CRE′ site (TGAGCTCA) near the transcription start site (TSS). D Mutation promoter assay for CREB-binding sites in the hRHOB gene (−118 to +44 bp). Plasmids without any promoter or enhancer were used as negative controls. Plasmids were constructed with wild-type (WT) CRE′, normal CRE (TGACGTCA) by reversing the middle GC, and random CRE (CCAAGGTT). TurboGFP expression was monitored in iHTMCs after NE stimulation. Dobutamine exposure (1 μM) for 4 h induced GFP expression (* p < 0.05, *** p < 0.001 vs. DMSO, one-way ANOVA [** p < 0.001], Dunnett’s multiple comparison), while random CRE had no effect ( p > 0.05). Values represent the means ± SEM of three independent experiments performed in triplicate. E NE and dobutamine induced RHOB expression (* p < 0.05, vs. DMSO, Dunnett’s multiple comparison test), but dexamethasone (Dex) did not. F Sp-cAMP and forskolin [FSK] with 3-isobutyl-1-methylxanthine [IBMX] also induced RHOB expression (* p < 0.05, vs. DMSO, Dunnett’s multiple comparison test). Data are presented as scatter plots with mean ± SEM (n = 3 independent experiments).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_79/10__1101_slash_2025__03__21__644679/10__1101_slash_2025__03__21__644679___F3.large.jpg)
![A RHOB knockout (KO) was achieved by transfection of CRISPR/Cas9 plasmid. B Western blot analysis of WT RHOB and RHOB-KO in iiHTMCs. C Cell growth of RHOB-KO iHTMC. KO had no effect on cell growth ( p > 0.05). D Effect of dobutamine on RHOB KO cell viability. Incubation with dobutamine for 6 h dose-dependently decreased cell viability (two-way ANOVA [concentration; * p = 0.0190]), whereas RHOB KO showed no effect. E Effect of 2 d of dobutamine treatment (0–10 μM) on TM aging. Low concentrations of dobutamine (10–100 nM) increased iHTMC senescence (* p < 0.05, *** p < 0.001, vs. DMSO, Dunnett’s multiple comparison test, two-way ANOVA [interaction, * p = 0.0122; time, ** p = 0.0073; coating, p < 0.2293]). F Effect of dobutamine on TM phagocytosis in RHOB KO cells. RHOB KO rescued dobutamine-suppressed TM phagocytosis (* p < 0.05, vs. WT, Šidák multiple comparison test, two-way ANOVA [concentration; * p = 0.0378, KO; ** p < 0.0012]). G Dobutamine gradually increased TM F-actin levels, but RHOB KO showed no effect (**** p < 0.0001 vs. DMSO, Dunnett’s multiple comparison test, two-way ANOVA [concentration, ** p = 0.0027; KO, p = 0.9508]). H Effect of dobutamine on TM aging in RHOB-KO cells. KO showed slightly decreased TM senescence at high dobutamine levels (** p < 0.01 vs. DMSO, Dunnett’s multiple comparison test, two-way ANOVA [interaction, * p = 0.0122; concentration; ** p = 0.0073; KO, p = 0.2293]). Data were normalized using signals from DMSO-treated cells and presented as bar graphs (mean ± SEM) with scatter plot of independent experiments (n = 3–4). I ROCK inhibitor Y27632 and RHO inhibitor rhosin rescued dobutamine-attenuated TM phagocytosis (* p < 0.05, vs. WT, Šidák multiple comparison test, two-way ANOVA [interaction, * p = 0.0342; concentration, ** p = 0.0030; KO, ** p = 0.0029]). J Lysophosphatidic acid (LPA), a RHO activator, dose-dependently attenuated TM phagocytosis (* p < 0.05 vs. DMSO, Dunnett’s multiple comparison test, one-way ANOVA [* p = 0.0202]). K Constitutively active (CA) RHOB (G14V in the GDP/GTP binding site) and RHOC WT structures. L Overexpression of RHOB WT and CA significantly inhibited phagocytosis, which was rescued by rhosin (* p < 0.05, vs. DMSO, Dunnett’s multiple comparison test, one-way ANOVA [* p = 0.0413]). Overexpression of WT RHOC had no effect on phagocytosis. N RHOB overexpression dramatically increased the number of adherent cells (*** p < 0.001 vs. DMSO, Dunnett’s multiple comparison test, one-way ANOVA [** p = 0.0002]). O RHOB overexpression inhibits liquid permeability in iHTMC multilayers (** p < 0.01, unpaired t-test). P Dobutamine-attenuated TM permeability was not observed in RHOB KO iHTMC multilayers (* p < 0.05, unpaired t -test).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_79/10__1101_slash_2025__03__21__644679/10__1101_slash_2025__03__21__644679___F5.large.jpg)
![A–C Effect of RHO-ROCK pathway-related inhibitors on nocturnal IOP increase in mice. Mice were administered Y27632 (100 µM/0.1% DMSO PBS), and rhosin (100 µM) at ZT10, and IOP was measured at ZT15. B Y27632 and rhosin treatment significantly suppressed the nocturnal IOP increase (paired t-test, *** p < 0.001). Data are presented as box-and-whisker plots of individual data (n = 6–16). C Inhibitors prevented IOP increase (* p < 0.05, **** p < 0.0001 vs. DMSO, one-way ANOVA [ p < 0.001], Dunnett’s multiple comparison test). Data are presented as scatter plots with mean ± SEM (n = 6–16). D,E Validation of the inhibitory effects of drugs on IOP using dobutamine (100 µM) treatment at ZT4. After 5 h, IOP was measured. Dobutamine significantly increased IOP, whereas pretreatment with Y27632 and rhosin prevented this effect (paired t-test, * p < 0.05, *** p < 0.001). F The same was true for individual data (*** p < 0.001, **** p < 0.0001 vs. Dobutamine+DMSO, one-way ANOVA [ p < 0.0001], Dunnett’s multiple comparison test). Data are presented as ( E ) box-and-whisker plots with individual data and ( F ) as scatter plots with mean ± SEM (n = 6–16). G Regulatory model of time-dependent systems by which nocturnal NE suppresses phagocytosis to induce AH outflow resistance through β1AR-cAMP-CREB-RHOB-ROCK activation, leading to an IOP increase at night.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_79/10__1101_slash_2025__03__21__644679/10__1101_slash_2025__03__21__644679___F6.large.jpg)