Estrogen and Cyp1b1 Regulate Pparγ in Pulmonary Hypertension Through a Ubiquitin‐Dependent Mechanism

Figures

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  Figure 1. Both 16αOHE and siRNA to CYP1B1 reduce Pparγ protein levels, but not mRNA levels. (A) Mouse pulmonary microvascular endothelial cells (mPMVEC) treated with 100 nM 16αOHE or 100 nM E2 for 24 h, 24 h after transfection with scrambled siRNA or siRNA to CYP1B1 lead to slight increases in PPARγ RNA expression, normalized to HPRT and to control. 16αOHE (B) mPMVEC with 24 h 100 nM 16aOHE treatment show reduction of PPARG protein of 30%–50%. This effect is abolished with 1 μM protease inhibitor MG132. (C) murine PMVEC treated with 100nm E2 or 16αOHE for 24 h, and scrambled siRNA or siRNA to CYP1B1, 24 h after transfection. (D) human primary PMVEC treated with 100 nM, E2 or 16αOHE for 24 h, and scrambled siRNA or siRNA to CYP1B1, 24 h after transfection.

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  Figure 2. siRNA to CYP1B1 synergizes with BMPR2 mutation to reduce fatty acid oxidation. (A) mPMVEC derived either from immortomice, or immortomice carrying the Bmpr2R899X transgene, 24 h after transfection with either scrambled siRNA or siRNA to Cyp1B1, and treated with either vehicle or 10 ng/ml BMP4 for 24 h, and probed for phosphorylated Smad1/5, the marker of canonical BMP pathway activity. (B) mPMVEC as above, probed for PPARγ, show reductions in protein with either CYP1B1 or Bmpr2R899X mutation, and additional reduction with both. (C) Baseline respiration rate (oxygen consumption) by Seahorse Oxygraphy with mPMVEC treated as above, using the fatty acid oxidation substrate palmitate‐BSA. (D) Maximum respiration rate in the above experiment. For both (C) and (D), each dot is an independent well. Error bars are standard error of mean, and statistical tests are two‐way ANOVA with Tukey‐Kramer HSD.

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  Figure 3. siCyp1B1 inhibition of Pparγ protein levels is through ubiquitination, but siCyp1b1 may also regulate activity through canonical Pparγ regulatory pathways. (A) Caspase inhibition (10μM caspase inhibitor zVAD‐FMK) in WT or Bmpr2 mutant PMVEC, with scrambled siRNA or siCyp1b1. siCyp1b1 reduces Pparγ levels, but this is not altered by caspase inhibition, in either WT or Bmpr2 mutant PVMEC. (B) Ubiquitin inhibition in WT or Bmpr2 mutant PMVEC, with scrambled siRNA or siCyp1b1. siCyp1b1 reduces Pparγ levels, but this is reversed with inhibition of ubiquitination. (C) immunoprecipitation with anti‐ubiquitin antibodies shows roughly doubling of pulldown of Pparγ with siCyp1b1 compared to scrambled siRNA. siCyp1b1 has no impact on IP with PPARγ and probe with Pparγ. (D) Phosphorylation of proteins associated with activation of Pparγ signaling have high congruence between murine and human PMVEC, with some signaling partners showing increased phosphorylation, some decreased, and some unchanged. our p‐JNK antibody didn't work for human samples. Two replicates are shown for each of human and mouse.

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  Figure 4. Noncanonical estrogen signaling effect on Pparγ protein levels are dependent on confluence and Bmpr2 mutation status. (A) Wild type and Bmpr2 mutant murine PMVEC at approximately 70% confluence are treated with cycloheximide and either vehicle or E2 bound to BSA, which cannot cross the cell membrane, and thus activates primarily noncanonical estrogen signaling. Numbers are densitometry, normalized to GAPDH. (B) Similar to (A), except using cells at approximately 95% confluence (most cells are completely confluent). (C) Ratio of PPARγ levels in E2‐BSA treated cells to that in controls, in (A) and (B). The numbers in the circles indicate which timepount they correspond to; the bars indicate average and standard deviation.

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  Figure 5. Impact of estrogen and Cyp1b1 siRNA are distinct in genes affected, but have substantial overlap in pathways affected. (A) Principal components analysis shows that the impact of estrogen or the estrogen metabolite 16αOHE is similar in cells with scrambled or siCyp1b1, but is along a nearly orthogonal vector to that separating scrambled siRNA from siCyp1b1. (B) Both estrogens and siCyp1b1 alter roughly a thousand transcript levels at p < 0.05 and fold change > 1.2, but with overlap in only about a quarter of the genes so changed in each group. (C) Significantly overrepresented gene ontology groups in cells with siCyp1b1 compared to control–gene lists are taken from (B). Radius corresponds to Log10(False Discovery Rate), angular width corresponds to number of genes in the group compared to total number of annotated genes with altered regulation. Overlap is because many genes fall into multiple ontology groups; degree of overlap is approximate because this is a two dimensional figure. Color is for clarity, and roughly groups ontology groups by overlap. Gray wedge at the bottom is differentially regulated genes which do not fit in any overregulated gene ontology group. (D) Similar to…

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  Figure 6. Cyp1b1 knockout mice have elevated RVSP, reduced Pparγ, and increased muscularization of small vessels. (A) Right ventricular systolic pressure (RVSP). Each circle is a measurement from one animal. All animals were fed western diet for nine weeks; the hypoxic mice were placed in normobaric 10% O2 for the last 3 weeks. Genotype effect p = 0.0015, hypoxia effect p < 0.0001, but interaction not significant by two way ANOVA. Pairwise comparisons using Tukey‐Kramer HSD. (B) Small vessel muscularization. Each circle is a measurement from one animal, which consists of counting ten 10× fields. Genotype effect p = 0.0002, hypoxia effect p < 0.0001, but interaction not significant by two way ANOVA. Pairwise comparisons using Tukey‐Kramer HSD. (C) Fulton index (RV/LV+S) is increased in hypoxic mice, but is not impact by Cyp1B1 knockout. (D) Weights of mice, normalized to their starting weight (starting weights were not different between groups). Cyp1B1−/− mice gained weight more slowly on high fat diet than wild‐type mice did, weight difference significant at every time point past 2 weeks. (E) Blood glucose at sacrifice–mice were not fasted. Lower levels in hypoxic WT animals is…

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  Figure 7. CYP1B1 knockout mice have alterations in genes related to lipid metabolism, cytokines, circadian rhythm, and vasoreactivity. For all parts, expression has been normalized to average expression of wild‐type mice. Wild type mice are represented by gray circles, and Cyp1B1−/− mice by black diamonds, with each symbol indicating expression in an individual animal. Average expression in Cyp1B1−/− mice is indicated by black horizontal bars with error bars, indicating SEM. All genes in each part fall within overrepresented gene ontology groups, with p values and gene‐ontology names at the top of each part.