AhR and CYP1B1 Control Oxygen Effects on Bone Marrow Progenitor Cells: The Enrichment of Multiple Olfactory Receptors as Potential Microbiome Sensors.

Polycyclic aromatic hydrocarbon (PAH) pollutants and microbiome products converge on the aryl hydrocarbon receptor (AhR) to redirect selective rapid adherence of isolated bone marrow (BM) cells. In young adult mice, Cyp1b1-deficiency and AhR activation by PAH, particularly when prolonged by Cyp1a1 deletion, produce matching gene stimulations in these BM cells. Vascular expression of Cyp1b1 lowers reactive oxygen species (ROS), suppressing NF-κB/RelA signaling. PAH and allelic selectivity support a non-canonical AhR participation, possibly through RelA. Genes stimulated by Cyp1b1 deficiency were further resolved according to the effects of Cyp1b1 and Cyp1a1 dual deletions (DKO). The adherent BM cells show a cluster of novel stimulations, including select developmental markers; multiple re-purposed olfactory receptors (OLFR); and α-Defensin, a microbial disruptor. Each one connects to an enhanced specific expression of the catalytic RNA Pol2 A subunit, among 12 different subunits. Mesenchymal progenitor BMS2 cells retain these features. Cyp1b1-deficiency removes lymphocytes from adherent assemblies as BM-derived mesenchymal stromal cells (BM-MSC) expand. Cyp1b1 effects were cell-type specific. In vivo, BM-MSC Cyp1b1 expression mediated PAH suppression of lymphocyte progenitors. In vitro, OP9-MSC sustained these progenitors, while Csf1 induced monocyte progenitor expansion to macrophages. Targeted Cyp1b1 deletion (Cdh5-Cre; Cyp1b1fl/fl) established endothelium control of ROS that directs AhR-mediated suppression of B cell progenitors. Monocyte Cyp1b1 deletion (Lyz2-Cre; Cyp1b1fl/fl) selectively attenuated M1 polarization of expanded macrophages, but did not enhance effects on basal M2 polarization. Thus, specific sources of Cyp1b1 link to AhR and to an OLFR network to provide BM inflammatory modulation via diverse microbiome products.

Cytochrome P4501B1 in bone marrow is co-expressed with key markers of mesenchymal stem cells. BMS2 cell line models PAH disruption of bone marrow niche development functions.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6 h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b > Tnfa> Ifng) within 6 h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.

Trace derivatives of kynurenine potently activate the aryl hydrocarbon receptor (AHR).

Cellular metabolites act as important signaling cues, but are subject to complex unknown chemistry. Kynurenine is a tryptophan metabolite that plays a crucial role in cancer and the immune system. Despite its atypical, non-ligand-like, highly polar structure, kynurenine activates the aryl hydrocarbon receptor (AHR), a PER, ARNT, SIM (PAS) family transcription factor that responds to diverse environmental and cellular ligands. The activity of kynurenine is increased 100-1000-fold by incubation or long-term storage and relies on the hydrophobic ligand-binding pocket of AHR, with identical structural signatures for AHR induction before and after activation. We purified trace-active derivatives of kynurenine and identified two novel, closely related condensation products, named trace-extended aromatic condensation products (TEACOPs), which are active at low picomolar levels. The synthesized compound for one of the predicted structures matched the purified compound in both chemical structure and AHR pharmacology. Our study provides evidence that kynurenine acts as an AHR pro-ligand, which requires novel chemical conversions to act as a receptor agonist.

Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism.

cAMP response element binding protein (CREB) is a key regulator of glucose metabolism and synaptic plasticity that is canonically regulated through recruitment of transcriptional coactivators. Here we show that phosphorylation of CREB on a conserved cluster of Ser residues (the ATM/CK cluster) by the DNA damage-activated protein kinase ataxia-telangiectasia-mutated (ATM) and casein kinase1 (CK1) and casein kinase2 (CK2) positively and negatively regulates CREB-mediated transcription in a signal dependent manner. In response to genotoxic stress, phosphorylation of the ATM/CK cluster inhibited CREB-mediated gene expression, DNA binding activity and chromatin occupancy proportional to the number of modified Ser residues. Paradoxically, substoichiometric, ATM-independent, phosphorylation of the ATM/CK cluster potentiated bursts in CREB-mediated transcription by promoting recruitment of the CREB coactivator, cAMP-regulated transcriptional coactivators (CRTC2). Livers from mice expressing a non-phosphorylatable CREB allele failed to attenuate gluconeogenic genes in response to DNA damage or fully activate the same genes in response to glucagon. We propose that phosphorylation-dependent regulation of DNA binding activity evolved as a tunable mechanism to control CREB transcriptional output and promote metabolic homeostasis in response to rapidly changing environmental conditions.

Mechanistic role of cytochrome P450 (CYP)1B1 in oxygen-mediated toxicity in pulmonary cells: A novel target for prevention of hyperoxic lung injury.

Supplemental oxygen, which is routinely administered to preterm infants with pulmonary insufficiency, contributes to bronchopulmonary dysplasia (BPD) in these infants. Hyperoxia also contributes to the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in adults. The mechanisms of oxygen-mediated pulmonary toxicity are not completely understood. Recent studies have suggested an important role for cytochrome P450 (CYP)1A1/1A2 in the protection against hyperoxic lung injury. The role of CYP1B1 in oxygen-mediated pulmonary toxicity has not been studied. In this investigation, we tested the hypothesis that CYP1B1 plays a mechanistic role in oxygen toxicity in pulmonary cells in vitro. In human bronchial epithelial cell line BEAS-2B, hyperoxic treatment for 1-3 days led to decreased cell viability by about 50-80%. Hyperoxic cytotoxicity was accompanied by an increase in levels of reactive oxygen species (ROS) by up to 110%, and an increase of TUNEL-positive cells by up to 4.8-fold. Western blot analysis showed hyperoxia to significantly down-regulate CYP1B1 protein level. Also, there was a decrease of CYP1B1 mRNA by up to 38% and Cyp1b1 promoter activity by up to 65%. On the other hand, CYP1B1 siRNA appeared to rescue the cell viability under hyperoxia stress, and overexpression of CYP1B1 significantly attenuated hyperoxic cytotoxicity after 48 h of incubation. In immortalized lung endothelial cells derived from Cyp1b1-null and wild-type mice, hyperoxia increased caspase 3/7 activities in a time-dependent manner, but endothelial cells lacking the Cyp1b1 gene showed significantly decreased caspase 3/7 activities after 48 and 72 h of incubation, implying that CYP1B1 might promote apoptosis in wild type lung endothelial cells under hyperoxic stress. In conclusion, our results support the hypothesis that CYP1B1 plays a mechanistic role in pulmonary oxygen toxicity, and CYP1B1-mediated apoptosis could be one of the mechanisms of oxygen toxicity. Thus, CYP1B1 could be a novel target for preventative and/or therapeutic interventions against BPD in infants and ALI/ARDS in adults.

Cyp1b1 affects external control of mouse hepatocytes, fatty acid homeostasis and signaling involving HNF4α and PPARα.

Cytochrome P450 1b1 (Cyp1b1) is expressed in endothelia, stellate cells and pre-adipocytes, but not hepatocytes. Deletion alters liver fatty acid metabolism and prevents obesity and hepatic steatosis. This suggests a novel extra-hepatocyte regulation directed from cells that express Cyp1b1. To characterize these mechanisms, microarray gene expression was analyzed in livers of normal and congenic Cyp1b1-ko C57BL/6 J mice fed either low or high fat diets. Cyp1b1-ko gene responses indicate suppression of endogenous PPARα activity, a switch from triglyceride storage to mitochondrial fatty acid oxidation and decreased oxidative stress. Many gene responses in Cyp1b1-ko are sexually dimorphic and correspond to increased activity of growth hormone mediated by HNF4α. Male responses stimulated by GH pulses are enhanced, whereas responses that decline exhibit further suppression, including Cyp regulation by PPARα, CAR and PXR. These effects of Cyp1b1 deletion overlap with effects caused by deletion of the small heterodimeric partner, a suppressor of these nuclear factors. Redirection of gene expression associated with liver fat homeostasis in Cyp1b1-ko mice that directs hypothalamic control of GH and leptin. Cyp1b1-ko suppresses neonatal Scd1 and delays adult maturation of dimorphic GH/HNF4α signaling. Alternatively, deletion may diminish hypothalamic metabolism of estradiol, which establishes adult GH regulation.

Cytochrome P450 1B1: An unexpected modulator of liver fatty acid homeostasis.

Cytochrome P450 1b1 (Cyp1b1) expression is absent in mouse hepatocytes, but present in liver endothelia and activated stellate cells. Increased expression during adipogenesis suggests a role of Cyp1b1 metabolism in fatty acid homeostasis. Wild-type C57BL/6j (WT) and Cyp1b1-null (Cyp1b1-ko) mice were provided low or high fat diets (LFD and HFD, respectively). Cyp1b1-deletion suppressed HFD-induced obesity, improved glucose tolerance and prevented liver steatosis. Suppression of lipid droplets in sinusoidal hepatocytes, concomitant with enhanced glycogen granules, was a consistent feature of Cyp1b1-ko mice. Cyp1b1 deletion altered the in vivo expression of 560 liver genes, including suppression of PPARγ, stearoyl CoA desaturase 1 (Scd1) and many genes stimulated by PPARα, each consistent with this switch in energy storage mechanism. Ligand activation of PPARα in Cyp1b1-ko mice by WY-14643 was, nevertheless, effective. Seventeen gene changes in Cyp1b1-ko mice correspond to mouse transgenic expression that attenuated diet-induced diabetes. The absence of Cyp1b1 in mouse hepatocytes indicates participation in energy homeostasis through extra-hepatocyte signaling. Extensive sexual dimorphism in hepatic gene expression suggests a developmental impact of estrogen metabolism by Cyp1b1. Suppression of Scd1 and increased leptin turnover support enhanced leptin participation from the hypothalamus. Cyp1b1-mediated effects on vascular cells may underlie these changes.

Lipidomics reveals a link between CYP1B1 and SCD1 in promoting obesity.

Cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of xenobiotic compounds and endogenous metabolites. Disruption of Cyp1b1 in mice results in suppression of high-fat diet (HFD)-induced obesity and an extensive change in hepatic energy regulation despite minimal constitutive expression of CYP1B1 in hepatocytes. Lack of CYP1B1 is correlated with altered lipid metabolism, especially lysophosphatidylcholines, contributing to protection against obesity. Ultraperformance liquid chromatography coupled to electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics revealed lysophosphatidylcholine 18:0 (LPC 18:0) as a biomarker positively related to HFD-induced obesity. The increased serum LPC 18:0 in wild-type mice is reduced in Cyp1b1-null mice on a HFD, which is reversed in CYP1B1-humanized mice. CYP1B1-humanized mice show higher diet-induced obesity compared with Cyp1b1-null mice, suggesting that human CYP1B1 shows a similar response to HFD as mouse Cyp1b1. In addition, hepatic stearoyl-CoA desaturase 1 (SCD1) expression was decreased in Cyp1b1-null mice, and the attenuated diet-induced obesity and lower serum LPC 18:0 in the Cyp1b1-null mice is elevated after SCD1 overexpression, suggesting that SCD1 is correlated with CYP1B1-induced obesity. These studies establish a biochemical link between cytochromes P450, lipids, and metabolic disorders and suggest that inhibition of CYP1B1 could be target for antiobesity drugs.

Lack of Cyp1b1 promotes the proliferative and migratory phenotype of perivascular supporting cells.

Perivascular supporting cells, including pericytes and smooth muscle cells (PC/SMC), have an integral role during angiogenesis and control vascular remodeling, maturation, and stabilization of neoteric vessels. We recently showed that a Cyp1B1 deficiency in mice results in the attenuation of angiogenesis in vivo and the pro-angiogenic activity of endothelial cells in vitro. However, the contribution of PC/SMC, and more specifically the cell autonomous effects of Cyp1B1 in these processes, needs further investigation. Here we demonstrate that PC constitutively expressed Cyp1B1, and that a deficiency in Cyp1B1 was associated with enhanced proliferation, and decreased apoptosis. Mechanistically, the lack of Cyp1B1 was associated with increased oxidative stress and sustained NF-κB activation, which was reversed by the antioxidant N-acetylcysteine. These changes were also concomitant with alterations in PC migration, adhesion, and expression of various extracellular matrix proteins, including thrombospondin-2. Cyp1B1-deficient PC also expressed decreased levels of vascular endothelial growth factor. Together, our results suggest an important role for Cyp1B1 expression in the regulation of PC proliferation, migration, and survival through modulation of the intracellular oxidative state and NF-κB expression and/or activity. Thus, a lack of Cyp1B1 in PC may have a significant role in vascular dysfunction and integrity, contributing to the attenuation of angiogenesis.

Stimulation of mouse Cyp1b1 during adipogenesis: characterization of promoter activation by the transcription factor Pax6.

Cytochrome P4501B1 (Cyp1b1) is expressed specifically in certain neural crest (NC) cells during embryogenesis. Mesenchymal progenitor cells that develop from NC cells are modeled here by mouse C3H10T1/2 and 3T3-L1 cells. Dexamethasone in combination with methylisobutylxanthine (DM) induces Cyp1b1 and a 6.7 kb mouse Cyp1b1 promoter-luciferase reporter in each cell type prior to adipogenesis. An 18 base sequence (at -6.11 kb) (PaxE) which was essential for this reporter stimulation in 3T3-L1 cells bound the transcription factor Pax6. This is shown by gel mobility shifts and sequence mutations. Heterologous vector expression of Pax6 in 3T3-L1 cells enhanced DM stimulated Cyp1b1 promoter activity through cooperation with two Sp1 sites in the proximal promoter region. Chromatin immunoprecipitation showed that DM stimulated binding of Pax6 adjacent to Sp1 in the proximal promoter more than in the PaxE region. The Cyp1b1 induction by DM in C3H10T1/2 cells was more rapid but independent of Pax6. The far upstream enhancer region (FUER) found in rat Cyp1b1 responded to DM but was inactive in the mouse promoter due to key sequence changes. The expression patterns of Pax6 and Cyp1b1 frequently overlap during mouse embryogenesis. The relationship between Pax6 and Cyp1b1 expression warrants further investigation, particularly in the NC.

Cytochrome P450 1B1: A Key Regulator of Ocular Iron Homeostasis and Oxidative Stress.

Cytochrome P450 (CYP) 1B1 belongs to the superfamily of heme-containing monooxygenases. Unlike other CYP enzymes, which are highly expressed in the liver, CYP1B1 is predominantly found in extrahepatic tissues, such as the brain, and ocular tissues including retina and trabecular meshwork. CYP1B1 metabolizes exogenous chemicals such as polycyclic aromatic hydrocarbons. CYP1B1 also metabolizes endogenous bioactive compounds including estradiol and arachidonic acid. These metabolites impact various cellular and physiological processes during development and pathological processes. We previously showed that CYP1B1 deficiency mitigates ischemia-mediated retinal neovascularization and drives the trabecular meshwork dysgenesis through increased levels of oxidative stress. However, the underlying mechanisms responsible for CYP1B1-deficiency-mediated increased oxidative stress remain largely unresolved. Iron is an essential element and utilized as a cofactor in a variety of enzymes. However, excess iron promotes the production of hydroxyl radicals, lipid peroxidation, increased oxidative stress, and cell damage. The retinal endothelium is recognized as a major component of the blood-retinal barrier, which controls ocular iron levels through the modulation of proteins involved in iron regulation present in retinal endothelial cells, as well as other ocular cell types including trabecular meshwork cells. We previously showed increased levels of reactive oxygen species and lipid peroxidation in the absence of CYP1B1, and in the retinal vasculature and trabecular meshwork, which was reversed by administration of antioxidant N-acetylcysteine. Here, we review the important role CYP1B1 expression and activity play in maintaining retinal redox homeostasis through the modulation of iron levels by retinal endothelial cells. The relationship between CYP1B1 expression and activity and iron levels has not been previously delineated. We review the potential significance of CYP1B1 expression, estrogen metabolism, and hepcidin-ferroportin regulatory axis in the local regulation of ocular iron levels.

Acute disruption of bone marrow hematopoiesis by benzo(a)pyrene is selectively reversed by aryl hydrocarbon receptor-mediated processes.

Bone marrow (BM) hematopoietic cells are selectively sensitive to polycyclic aromatic hydrocarbons (PAH) in vivo. 7,12-Dimethylbenz(a)anthracene (DMBA), but not benzo(a)pyrene (BP), depletes BM hematopoietic cells in C57BL/6 mice. This difference is due to a BP-selective aryl hydrocarbon receptor (AhR)-mediated recovery. Colony-forming unit assays show suppression of lymphoid progenitors by each PAH within 6 h but a subsequent recovery, exclusively after BP treatment. Suppression of myeloid progenitors (6 h) occurs only for DMBA. Each progenitor responded equally to DMBA and BP in congenic mice expressing the PAH-resistant AhR (AhR(d)). AhR, therefore, mediates this BP recovery in each progenitor type. These PAH suppressions depend on Cyp1b1-mediated metabolism. Paradoxically, few genes responded to DMBA, whereas 12 times more responded to BP. Progenitor suppression by DMBA, therefore, occurs with minimal effects on the general BM population. Standard AhR-mediated stimulations (Cyp1a1, Cyp1b1, Ahrr) were similar for each PAH and for the specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin but were absent in AhR(d) mice. A group of 12 such AhR responses was sustained from 6 to 24 h. A second, larger set of BP responses (chemokines, cytokines, cyclooxygenase 2) differed in two respects; DMBA responses were low and BP responses declined extensively from 6 to 24 h. A third cluster exhibited BP-induced increases in protective genes (Nqo1, GST-mu) that appeared only after 12 h. Conversion of BP to quinones contributes oxidative signaling not seen with DMBA. We propose that genes in this second cluster, which share oxidative signaling and AhR activation, provide the AhR-dependent protection of hematopoietic progenitors seen for BP.

CYP1B1 expression promotes the proangiogenic phenotype of endothelium through decreased intracellular oxidative stress and thrombospondin-2 expression.

Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1(-/-)) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1(-/-) mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1(-/-) ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1(-/-) mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1(-/-) ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1(-/-) ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization.

Cholesterol Contributes to Male Sex Differentiation Through Its Developmental Role in Androgen Synthesis and Hedgehog Signaling.

Two specialized functions of cholesterol during fetal development include serving as a precursor to androgen synthesis and supporting hedgehog (HH) signaling activity. Androgens are produced by the testes to facilitate masculinization of the fetus. Recent evidence shows that intricate interactions between the HH and androgen signaling pathways are required for optimal male sex differentiation and defects of either can cause birth anomalies indicative of 46,XY male variations of sex development (VSD). Further, perturbations in cholesterol synthesis can cause developmental defects, including VSD, that phenocopy those caused by disrupted androgen or HH signaling, highlighting the functional role of cholesterol in promoting male sex differentiation. In this review, we focus on the role of cholesterol in systemic androgen and local HH signaling events during fetal masculinization and their collective contributions to pediatric VSD.

Distinctive functioning of STARD1 in the fetal Leydig cells compared to adult Leydig and adrenal cells. Impact of Hedgehog signaling via the primary cilium.

STARD1 stimulates cholesterol transfer to mitochondrial CYP11A1 for conversion to pregnenolone. A cholesterol-binding START domain is guided by an N-terminal domain in a cell selective manner. Fetal and adult Leydig cells (FLC, ALC) show distinct Stard1 regulation. sm- FISH microscopy, which resolves individual molecules of Stard1 mRNA, shows uniformly high basal expression in each FLC. In ALC, in vivo, and cultured MA-10 cells, basal Stard1 expression is minimal. PKA activates loci asynchronously, with delayed splicing/export of 3.5 kb mRNA to mitochondria. After 60 min, ALC transition to an integrated mRNA delivery to mitochondria that is seen in FLC. Sertoli cells cooperate in Stard1 stimulation in FLC by delivering DHH to the primary cilium. There PTCH, SMO and cholesterol cooperate to release GLI3 to activate the Stard1 locus, probably by directing histone changes. ALC lack cilia. PKA then primes locus activation. FLC and ALC share similar SIK/CRTC/CREB regulation characterized for adrenal cells.

CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress.

We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.

STARD1 Functions in Mitochondrial Cholesterol Metabolism and Nascent HDL Formation. Gene Expression and Molecular mRNA Imaging Show Novel Splicing and a 1:1 Mitochondrial Association.

STARD1 moves cholesterol (CHOL) from the outer mitochondrial membrane (OMM) to the inner membrane (IMM) in steroidogenic cells. This activity is integrated into CHOL trafficking and synthesis homeostasis, involving uptake through SR-B1 and LDL receptors and distribution through endosomes, ER, and lipid droplets. In adrenal cells, STARD1 is imported into the mitochondrial matrix accompanied by delivery of several hundred CHOL molecules. This transfer limits CYP11A1-mediated generation of pregnenolone. CHOL transfer is coupled to translation of STARD1 mRNA at the OMM. In testis cells, slower CHOL trafficking seems to be limiting. STARD1 also functions in a slower process through ER OMM contacts. The START domain of STARD1 is utilized by a family of genes, which includes additional STARD (forms 3-6) and GRAMD1B proteins that transfer CHOL. STARD forms 2 and 7 deliver phosphatidylcholine. STARD1 and STARD7 target their respective activities to mitochondria, via N-terminal domains (NTD) of over 50 amino acids. The NTD is not essential for steroidogenesis but exerts tissue-selective enhancement (testis>>adrenal). Three conserved sites for cleavage by the mitochondrial processing protease (MPP) generate three forms, each potentially with specific functions, as demonstrated in STARD7. STARD1 is expressed in macrophage and cardiac repair fibroblasts. Additional functions include CHOL metabolism by CYP27A1 that directs activation of LXR and CHOL export processes. STARD1 generates 3.5- and 1.6-kb mRNA from alternative polyadenylation. The 3.5-kb form exclusively binds the PKA-induced regulator, TIS11b, which binds at conserved sites in the extended 3'UTR to control mRNA translation and turnover. STARD1 expression also exhibits a novel, slow splicing that delayed splicing delivery of mRNA to mitochondria. Stimulation of transcription by PKA is directed by suppression of SIK forms that activate a CRTC/CREB/CBP promoter complex. This process is critical to pulsatile hormonal activation in vivo. sm-FISH RNA imaging shows a flow of single STARD1 mRNA particles from asymmetric accumulations of primary transcripts at gene loci to 1:1 complex of 3.5-kb mRNA with peri-nuclear mitochondria. Adrenal cells are similar but distinguished from testis cells by appreciable basal expression prior to hormonal activation. This difference is conserved in culture and in vivo.

Cyp1b1 directs Srebp-mediated cholesterol and retinoid synthesis in perinatal liver; Association with retinoic acid activity during fetal development.

BACKGROUND: Cytochrome P450 1b1 (Cyp1b1) deletion and dietary retinol deficiency during pregnancy (GVAD) affect perinatal liver functions regulated by Srebp. Cyp1b1 is not expressed in perinatal liver but appears in the E9.5 embryo, close to sites of retinoic acid (RA) signaling. HYPOTHESIS: Parallel effects of Cyp1b1 and retinol on postnatal Srebp derive from effects in the developing liver or systemic signaling. APPROACH: Cluster postnatal increases in hepatic genes in relation to effects of GVAD or Cyp1b1 deletion. Sort expression changes in relation to genes regulated by Srebp1 and Srebp2.Test these treatments on embryos at E9.5, examining changes at the site of liver initiation. Use in situ hybridization to resolve effects on mRNA distributions of Aldh1a2 and Cyp26a1 (RA homeostasis); Hoxb1 and Pax6 (RA targets). Assess mice lacking Lrat and Rbp4 (DKO mice) that severely limits retinol supply to embryos. RESULTS: At birth, GVAD and Cyp1b1 deletion stimulate gene markers of hepatic stellate cell (HSC) activation but also suppress Hamp. These treatments then selectively prevent the postnatal onset of genes that synthesize cholesterol (Hmgcr, Sqle) and fatty acids (Fasn, Scd1), but also direct cholesterol transport (Ldlr, Pcsk9, Stard4) and retinoid synthesis (Aldh1a1, Rdh11). Extensive support by Cyp1b1 is implicated, but with distinct GVAD interventions for Srebp1 and Srebp2. At E9.5, Cyp1b1 is expressed in the septum transversum mesenchyme (STM) with ß-carotene oxygenase (Bco1) that generates retinaldehyde. STM provides progenitors for the HSC and supports liver expansion. GVAD and Cyp1b1-/- do not affect RA-dependent Hoxb1 and Pax6. In DKO embryos, RA-dependent Cyp26a1 is lost but Hoxb1 is sustained with Cyp1b1 at multiple sites. CONCLUSION: Cyp1b1-/- suppresses genes supported by Srebp. GVAD effects distinguish Srebp1 and Srebp2 mediation. Srebp regulation overlaps appreciably in cholesterol and retinoid homeostasis. Bco1/Cyp1b1 partnership in the STM may contribute to this later liver regulation.

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

Liver X receptor opens a new gateway to StAR and to steroid hormones.

Liver X receptors (LXRs) broadly limit cholesterol accumulation by regulating expression of genes involved in cholesterol efflux and storage. In this issue of the JCI, Cummins et al. report that LXRalpha is involved in similar regulation in the adrenal cortex, but it also substantially modulates glucocorticoid synthesis (see the related article beginning on page 1902). LXRalpha deletion in mice increases the availability of adrenal cholesterol for steroid synthesis by decreasing the expression of cholesterol efflux transporters. Glucocorticoid synthesis requires intramitochondrial cholesterol transport mediated by the steroidogenic acute regulatory protein (StAR). Surprisingly, LXR deletion and stimulation by an agonist each increase glucocorticoid synthesis. This parallels increased expression of StAR and several other steroidogenic genes.