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Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline variants in the diacylglycerol (DAG)/Ca2+-regulated protein kinase Cγ (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Most of the identified mutations cluster in the DAG-sensing C1 domains. Here, we found with a FRET-based activity reporter that SCA14-associated PKCγ mutations, including a previously undescribed variant, D115Y, enhanced the basal activity of the kinase by compromising its autoinhibition. Unlike other mutations in PKC that impair its autoinhibition but lead to its degradation, the C1 domain mutations protected PKCγ from such down-regulation. This enhanced basal signaling rewired the brain phosphoproteome, as revealed by phosphoproteomic analysis of cerebella from mice expressing a human SCA14-associated H101Y mutant PKCγ transgene. Mutations that induced a high basal activity in vitro were associated with earlier average age of onset in patients. Furthermore, the extent of disrupted autoinhibition, but not agonist-stimulated activity, correlated with disease severity. Molecular modeling indicated that almost all SCA14 variants not within the C1 domain were located at interfaces with the C1B domain, suggesting that mutations in and proximal to the C1B domain are a susceptibility for SCA14 because they uniquely enhance PKCγ basal activity while protecting the enzyme from down-regulation. These results provide insight into how PKCγ activation is modulated and how deregulation of the cerebellar phosphoproteome by SCA14-associated mutations affects disease progression.

The aims of the present study were to determine uterine, vaginal and placental blood flows by Doppler ultrasound cross-buffalo gestation and to evaluate the relationships among reproductive Doppler parameters and serum metabolic parameters as well as oxidative stress. Uterine (UA) and vaginal (VA) arteries were scanned every month, and placentome was scanned from month 4 till 8 in gestation. Time-averaged maximum velocity (TAMV), pulsatility index (PI), resistance index (RI), systolic/diastolic ratio (SD) and arterial diameter (AD) were used for accessing UA and VA hemodynamics. Time-averaged maximum velocity positively correlated with and AD, and both negatively correlated with their PI, RI and SD in UA and VA. TAMV and AD increased constantly in pregnancy, with maximum increase in months 4 and 9. Pulsatility index, RI and AD of UA decreased between months 4 and 9, while PI, RI and AD of VA decreased between months 5 and 9 and then increased in month 10 in pregnancy. Time-averaged maximum velocity of placentome blood flow increased exponentially from months 4 to 8, but decreased at the last two months in pregnancy. Serum lipids were significantly higher in the first month compared to all other months, while glucose was significantly lower in months 9 and 10. Malondialdehyde increased from month 3 till term, but peaked in month 5 and 10. Glutathione and catalase were highest in the first month and remained after. Time-averaged maximum velocity and AD for both UA and VA negatively correlated with serum lipids, glucose, catalase and glutathione, while positively correlated with malondialdehyde and total protein. Thus, increases in uterine blood flow (UtBF), vaginal blood flow (VaBF) and placental blood flow (PaBF) are associated with increased metabolism and oxidative stress in buffalo pregnancy.

Dysregulated immune hemostasis occurs in unexplained recurrent spontaneous abortion (URSA). Synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), hydrogen sulfide (H2S) promotes regulatory T-cell differentiation and regulates immune hemostasis; yet, its role in URSA is elusive. To determine if H2S plays a role in early pregnancy and if dysregulated H2S signaling results in recurrent spontaneous abortion. First trimester placenta villi and decidua were collected from normal and URSA pregnancies. Protein expression was examined by immunohistochemistry and immunoblotting. Human trophoblast HTR8/SVneo and JEG3 cells were treated with H2S donors; HTR8/SVneo cells were transfected with CBS ribonucleic acid interference (RNAi) or complementary deoxyribonucleic acid. Cell migration and invasion were determined by transwell assays; trophoblast transcriptomes were determined by RNA sequencing (RNA-seq). Wild-type, CBS-deficient, and CBA/J × DBA/2 mice were treated with CBS and CSE inhibitors or H2S donors to determine the role of H2S in early pregnancy in vivo. CBS and CSE proteins showed cell-specific expressions, but only CBS decreased in the villous cytotrophoblast in URSA versus normal participants. H2S donors promoted migration and invasion and MMP-2 and VEGF expression in human placenta trophoblast cells that contain SV40 viral deoxyribonucleic acid sequences (HTR8/SVneo) and human placenta trophoblast cells (JEG3 cells), similar to forced CBS expression in HTR8/SVneo cells. The CBS-responsive transcriptomes in HTR8/SVneo cells contained differentially regulated genes (ie, interleukin-1 receptor and prostaglandin-endoperoxide synthase 2) that are associated with nuclear factor-κB-mediated inflammatory response. In vivo, dysregulated CBS/H2S signaling significantly increased embryonic resorption and decidual T-helper 1/T-helper 2 imbalance in mice, which was partially rescued by H2S donors. CBS/H2S signaling maintains early pregnancy, possibly via regulating maternal-fetal interface immune hemostasis, offering opportunities for H2S-based immunotherapies for URSA.

The pregnancy-augmented uterine vasodilation is linked to increased AT2R (angiotensin type-2 receptor) that mediates the vasodilatory effects of angiotensin II. However, the mechanisms controlling AT2R expression during pregnancy remain unclear. Estrogens are known to play a role in vascular adaptations during pregnancy. We hypothesized that estrogen stimulates uterine artery AT2R expression via ER (estrogen receptor)-β-dependent transcription in a pregnancy-specific endothelium-dependent manner. Plasma estradiol levels increased and peaked in late pregnancy and returned to prepregnant levels post-partum, correlating with uterine artery AT2R and ERβ upregulation. Estradiol stimulated AT2R mRNA expression in endothelium-intact but not endothelium-denuded late pregnant and nonpregnant rat uterine artery ex vivo. Consistently, estradiol stimulated AT2R mRNA expression in late pregnant but not nonpregnant primary human uterine artery endothelial cells in vitro, which was abolished by ER antagonist ICI 182,780. Higher ERα protein bound to ER-responsive elements in AT2R promoter in the nonpregnant arteries whereas higher ERβ bound in the pregnant state. ERα protein levels were similar but higher ERβ protein levels were expressed in pregnant versus nonpregnant human uterine artery endothelial cells. Estradiol stimulation recruited ERα to the AT2R promoter in the nonpregnant state and ERβ to the AT2R promoter in pregnancy; however, only ERβ recruitment mediated transactivation of the AT2R reporter gene in pregnant human uterine artery endothelial cells. Estradiol-induced AT2R expression was abolished by the specific ERβ (not ERα) antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) and mimicked by the specific ERβ (not ERα) agonist 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) in pregnant human uterine artery endothelial cells in vitro. This study demonstrates a novel role of pregnancy-augmented ERβ in AT2R upregulation in the uterine artery and provides new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

Organs are sculpted by extracellular as well as cell-intrinsic forces, but how collective cell dynamics are orchestrated in response to environmental cues is poorly understood. Here we apply advanced image analysis to reveal extracellular matrix-responsive cell behaviors that drive elongation of the Drosophila follicle, a model system in which basement membrane stiffness instructs three-dimensional tissue morphogenesis. Through in toto morphometric analyses of wild type and round egg mutants, we find that neither changes in average cell shape nor oriented cell division are required for appropriate organ shape. Instead, a major element is the reorientation of elongated cells at the follicle anterior. Polarized reorientation is regulated by mechanical cues from the basement membrane, which are transduced by the Src tyrosine kinase to alter junctional E-cadherin trafficking. This mechanosensitive cellular behavior represents a conserved mechanism that can elongate edgeless tubular epithelia in a process distinct from those that elongate bounded, planar epithelia.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl-/H+ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and -genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term ≈ 145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P > > > NP follicular > luteal UAendo and UAvsm (P  > > NP UAendo and UAvsm (P

Endogenous hydrogen sulfide (H2 S), synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), is a potent vasodilator that can be stimulated by estradiol-17β (E 2 β) in uterine artery (UA) smooth muscle (UASMC) in vivo; however, the underlying mechanisms are unknown. This study tested a hypothesis that E 2 β stimulates H 2 S biosynthesis by upregulating CBS expression via specific estrogen receptor (ER). Treatment with E 2 β stimulated time- and concentration- dependent CBS and CSE messenger RNA (mRNA) and protein expressions, and H 2 S production in cultured primary UASMC isolated from late pregnant ewes, which were blocked by ICI 182,780. Treatment with specific ERα or ERβ agonist mimicked these E 2 β-stimulated responses, which were blocked by specific ERα or ERβ antagonist. Moreover, E 2 β activated both CBS and CSE promoters and ICI 182,780 blocked the E 2 β-stimulated responses. Thus, E 2 β stimulates H 2 S production by upregulating CBS and CSE expression via specific ER-dependent transcription in UASMC in vitro.

Pregnancy is a physiologic state of substantially elevated estrogen biosynthesis that maintains vasodilator production by uterine artery endothelial cells (P-UAECs) and thus uterine perfusion. Estrogen receptors (ER-α and ER-β; ESR1 and ESR2) stimulate nongenomic rapid vasodilatory responses partly through activation of endothelial nitric oxide synthase (eNOS). Rapid estrogenic responses are initiated by the ∼4% ESRs localized to the plasmalemma of endothelial cells. Caveolin-1 (Cav-1) interactions within the caveolae are theorized to influence estrogenic effects mediated by both ESRs. Hypothesis: Both ESR1 and ESR2 display similar spatial partitioning between the plasmalemma and nucleus of UAECs and have similar interactions with Cav-1 at the plasmalemma. Using transmission electron microscopy, we observed numerous caveolae structures in UAECs, while immunogold labeling and subcellular fractionations identified ESR1 and ESR2 in three subcellular locations: membrane, cytosol, and nucleus. Bioinformatics approaches to analyze ESR1 and ESR2 transmembrane domains identified no regions that facilitate ESR interaction with plasmalemma. However, sucrose density centrifugation and Cav-1 immunoisolation columns uniquely demonstrated very high protein-protein association only between ESR1, but not ESR2, with Cav-1. These data demonstrate (1) both ESRs localize to the plasmalemma, cytosol and nucleus; (2) neither ESR1 nor ESR2 contain a classic region that crosses the plasmalemma to facilitate attachment; and (3) ESR1, but not ESR2, can be detected in the caveolar subcellular domain demonstrating ESR1 is the only ESR bound in close proximity to Cav-1 and eNOS within this microdomain. Lack of protein-protein interaction between Cav-1 and ESR2 demonstrates a novel independent association of these proteins at the plasmalemma.

Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). Estrogens are potent vasodilators that stimulate H2S biosynthesis in uterine arteries (UA) in vivo; however, the underlying mechanisms are unknown. We hypothesized that estrogens stimulate H2S biosynthesis in UA endothelial cells (UAEC) via specific estrogen receptor (ER)-dependent mechanisms. In cultured primary UAEC, treatment with estradiol-17β (E2β) stimulated CBS and CTH mRNAs and proteins in a time- and concentration-dependent fashion. As little as 0.1 nM E2β was effective in increasing CBS and CTH expressions and these stimulatory effects maximized with 10-100 nM E2β at 48-72 h. E2β also activated CBS and CTH promoters in UAEC, leading to CBS and CTH expression. Treatment with E2β stimulated H2S production, which was blocked by specific inhibitors of either CBS or CTH and their combination and the ER antagonist ICI 182780. Treatment with either specific agonist of ERα or ERβ stimulated both CBS and CTH mRNA and protein expressions and H2S production to levels similar to that of E2β. Specific antagonist of either ERα or ERβ blocked E2β-stimulated CBS and CTH mRNA and protein expressions and H2S production. Combinations of either ERα or ERβ agonists or their antagonists had no additive effects. Thus, E2β stimulates H2S production by upregulating CBS and CTH mRNA and protein expressions through specific ERα or ERβ-dependent CBS and CTH transcription in UAEC in vitro.

Experimental evidence shows that parental psychological stress affects the long-term health of offspring in an inheritable fashion. Although epigenetic mechanisms, including DNA methylation, miRNA, and histone modifications, are involved in transgenerational programming, the underlining mechanisms of transgenerational inheritance remain unsolved. Here, we present a single-cell-based computational model for transgenerational inheritance for investigating the long-term dynamics of phenotype changes in response to parental stress. The model is based on a recent study that has identified the imprinted sperm gene Sfmbt2 as a key target, and incorporates crosstalks among drastically different time scales in mammalian development, including DNA methylation, transcription, cell division, and population dynamics. Computational analysis of the model suggests a positive feedback to DNA methylation in the promoter region of sperm Sfmbt2 gene that provides a possible mechanism to mediate the parental psychological stress reprogramming in offspring. This approach provides a modeling framework for the understanding of the roles that epigenetics play in transgenerational inheritance.

Cell migration is indispensable to morphogenesis and homeostasis. Live imaging allows mechanistic insights, but long-term observation can alter normal biology, and tools to track movements in vivo without perturbation are lacking. We develop here a tool called M-TRAIL (matrix-labeling technique for real-time and inferred location), which reveals migration histories in fixed tissues. Using clones that overexpress GFP-tagged extracellular matrix (ECM) components, motility trajectories are mapped based on durable traces deposited onto basement membrane. We applied M-TRAIL to Drosophila follicle rotation, comparing in vivo and ex vivo migratory dynamics. The rate, trajectory, and cessation of rotation in wild-type (WT) follicles measured in vivo and ex vivo were identical, as was rotation failure in fat2 mutants. However, follicles carrying intracellularly truncated Fat2, previously reported to lack rotation ex vivo, in fact rotate in vivo at a reduced speed, thus revalidating the hypothesis that rotation is required for tissue elongation. The M-TRAIL approach could be applied to track and quantitate in vivo cell motility in other tissues and organisms.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1.

Long non-coding RNAs (lncRNAs) play an important role in gene regulation and are involving in diverse cellular processes. However, their roles in reprogramming of gene expression profiles during lineage commitment and maturation of mesenchymal stem cells (MSCs) remain poorly understood. In the current study, we characterize the expression of a lncRNA, HoxA-AS3, during the differentiation of MSCs. We showed that HoxA-AS3 is increased upon adipogenic induction of MSCs, while HoxA-AS3 remains unaltered during osteogenic induction. Silencing of HoxA-AS3 in MSCs resulted in decreased adipogenesis and expression of adipogenic markers, PPARG, CEBPA, FABP4 and ADIPOQ. Conversely, knockdown of HoxA-AS3 expression in MSCs exhibited an enhanced osteogenesis and osteogenic markers expression, including RUNX2, SP7, COL1A1, IBSP, BGLAP and SPP1. Mechanistically, HoxA-AS3 interacts with Enhancer Of Zeste 2 (EZH2) and is required for H3 lysine-27 trimethylation (H3K27me3) of key osteogenic transcription factor Runx2. Our data reveal that HoxA-AS3 acts as an epigenetic switch that determines the lineage specification of MSC.

Planar cell polarity (PCP) information is a critical determinant of organ morphogenesis. While PCP in bounded epithelial sheets is increasingly well understood, how PCP is organized in tubular and acinar tissues is not. Drosophila egg chambers (follicles) are an acinus-like edgeless epithelium and exhibit a continuous, circumferential PCP that does not depend on pathways active in bounded epithelia; this follicle PCP directs formation of an ellipsoid rather than a spherical egg. Here, we apply an imaging algorithm to unroll the entire 3D tissue surface and comprehensively analyze PCP onset. This approach traces chiral symmetry breaking to plus-end polarity of microtubules in the germarium, well before follicles form and rotate. PCP germarial microtubules provide chiral information that predicts the direction of whole-tissue rotation as soon as independent follicles form. Concordant microtubule polarity, but not microtubule alignment, requires the atypical cadherin Fat2, which acts at an early stage to translate plus-end bias into coordinated actin-mediated collective cell migration. Because microtubules are not required for PCP or migration after follicle rotation initiates, while dynamic actin and extracellular matrix are, polarized microtubules lie at the beginning of a handoff mechanism that passes early chiral PCP of the cytoskeleton to a supracellular planar polarized extracellular matrix and elongates the organ.

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis.

Estrogens dramatically dilate numerous vascular beds with the greatest response in the uterus. Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger, which is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We hypothesized that estrogen replacement therapy (ERT) selectively stimulates H2S biosynthesis in uterine artery (UA) and other systemic arteries. Intact and endothelium-denuded UA, mesenteric artery (MA), and carotid artery (CA) were obtained from ovariectomized nonpregnant ewes (n = 5/group) receiving vehicle or estradiol-17β replacement therapy (ERT). Total RNA and protein were extracted for measuring CBS and CSE, and H2S production was determined by the methylene blue assay. Paraffin-embedded UA rings were used to localize CBS and CSE proteins by immunofluorescence microscopy. ERT significantly stimulated CBS mRNA and protein without altering CSE mRNA or protein in intact and denuded UA. Quantitative immunofluorescence microscopic analyses showed CBS and CSE protein localization in endothelium and smooth muscle and confirmed that ERT stimulated CBS but not CSE protein expression in UA endothelium and smooth muscle. ERT also stimulated CBS, but not CSE, mRNA and protein expression in intact and denuded MA but not CA in ovariectomized ewes. Concomitantly, ERT stimulated UA and MA but not CA H2S production. ERT-stimulated UA H2S production was completely blocked by a specific CBS but not CSE inhibitor. Thus, ERT selectively stimulates UA and MA but not CA H2S biosynthesis by specifically up-regulating CBS expression, implicating a role of H2S in estrogen-induced vasodilation and postmenopausal women's health.

The induction of autophagy in the mammalian heart during the perinatal period is an essential adaptation required to survive early neonatal starvation; however, the mechanisms that mediate autophagy suppression once feeding is established are not known. Insulin signaling in the heart is transduced via insulin and IGF-1 receptors (IGF-1Rs). We disrupted insulin and IGF-1R signaling by generating mice with combined cardiomyocyte-specific deletion of Irs1 and Irs2. Here we show that loss of IRS signaling prevented the physiological suppression of autophagy that normally parallels the postnatal increase in circulating insulin. This resulted in unrestrained autophagy in cardiomyocytes, which contributed to myocyte loss, heart failure, and premature death. This process was ameliorated either by activation of mTOR with aa supplementation or by genetic suppression of autophagic activation. Loss of IRS1 and IRS2 signaling also increased apoptosis and precipitated mitochondrial dysfunction, which were not reduced when autophagic flux was normalized. Together, these data indicate that in addition to prosurvival signaling, insulin action in early life mediates the physiological postnatal suppression of autophagy, thereby linking nutrient sensing to postnatal cardiac development.

Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-β-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis.

ATP leads to endothelial NO synthase (eNOS)/NO-mediated vasodilation, a process hypothesized to depend on the endothelial caveolar eNOS partitioning and subcellular domain-specific multisite phosphorylation state. We demonstrate herein that, in both the absence and presence of ATP, the uterine artery endothelial caveolae contain specific protein machinery related to subcellular partitioning and act as specific focal "hubs" for NO- and ATP-related proteins. ATP-induced eNOS regulation showed a complex set of multisite posttranslational phosphorylation events that were closely associated with the enzyme's partitioning between caveolar and noncaveolar endothelial subcellular domains. The comprehensive model that we present demonstrates that ATP repartitioned eNOS between the caveolar and noncaveolar subcellular domains; specifically, the stimulatory (PSer635)eNOS was substantially higher in the caveolar pool with subcellular domain-independent increased levels on ATP treatment. The stimulatory (PSer1179)eNOS was not altered by ATP treatment. However, the inhibitory (PThr495)eNOS was regulated predominantly in the caveolar domain with decreased levels on ATP action. In contrast, the agonist-specific (PSer114)eNOS was localized in the noncaveolar pool with increased levels on ATP stimulation. Thus, the endothelial caveolar membrane system plays a pivotal role(s) in ATP-associated subcellular partitioning and possesses the relevant protein machinery for ATP-induced NO regulation. Furthermore, these subcellular domain-specific phosphorylation/dephosphorylation events provide evidence relating to eNOS spatio-temporal dynamics.

Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10-30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro-L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two-dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, nine were novel SNO-proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation.

Vascular endothelial growth factor (VEGF) is one of the best characterized angiogenic factors controlling placental angiogenesis; however, how VEGF regulates placental angiogenesis has not yet completely understood. In this study, we found that all the components of assembling a functional NADPH oxidase (NOX2, p22(phox), p47(phox), p67(phox), and Rac1) are expressed in ovine fetoplacental artery endothelial cells (oFPAECs) in vitro and ex vivo. Treatment with VEGF (10 ng/ml) rapidly and transiently activated Rac1 in oFPAECs in vitro and increased Rac1 association with p67(phox) in 5 min. Intracellular superoxide formation began to significantly increase after 25-30 min of VEGF stimulation, which was mediated by both VEGFR1 and VEGFR2. VEGF also stimulated oFPAE cell proliferation and migration and enhanced the formation of tube-like structures on Matrigel matrix. In oFAPEC transfected with specific Rac1 small interfering RNA (siRNA, 40 nm), VEGF-induced intracellular superoxide formation was completely abrogated in association with a 78% reduction of endogenous Rac1. In oFPAE cells transfected with the specific Rac1 siRNA, but not with transfection reagent alone or scrambled control siRNA, VEGF-induced cell proliferation, migration, and tube-like structure formation were dramatically inhibited. Pretreatment of an NADPH oxidase inhibitor apocynin also abrogates the VEGF-stimulated intracellular superoxide production and DNA synthesis in oFPAECs. Taken together, our results demonstrated that a Rac1/Nox2-based NADPH oxidase system is present in placental endothelial cells. This NADPH oxidase system appears to generate the second messenger superoxide that plays a critical role in the signaling control of the VEGF-induced placental angiogenesis.

Currently, no single marker is sensitive and specific enough to be considered a reliable biomarker for prenatal alcohol exposure. To identify a proteomic signature profile for maternal alcohol consumption, we carried out high-throughput proteomics on maternal endothelial caveolae exposed to moderate binge-like alcohol conditions. In these specialized lipid-ordered microdomains that contain a rich assembly of proteins, we demonstrate that moderate binge-like alcohol resulted in a distinctive maternal caveolar proteomic signature with important proteins being dramatically decreased/knocked out in the alcoholic profile. These proteins span from histones and basic structural proteins like α tubulin to proteins involved in trafficking, deubiquitination, cell signaling, and cell-cell adhesion. The profile also suggests an important role for the mother and the uteroplacental compartment in the pathogenesis of fetal alcohol spectrum disorders (FASD). These data demonstrate that the caveolar proteomic signature created by alcohol shows a promising direction for early detection of FASD.

A novel family of evolutionally conserved neuronal guidance cues, including ligands (i.e., Slit, netrin, epherin, and semaphorin) and their corresponding receptors (i.e., Robo, DCC/Unc5, Eph and plexin/ neuropilin), has been identified to play a crucial role in axon pathfinding and branching as well as neuronal cell migration. The presence of commonalities in both neural and vascular developments has led to some exciting discoveries recently, which have extended the functions of these systems to vascular formation (vasculogenesis) and development (angiogenesis). Some of these ligands and receptors have been found to be expressed in the vasculature and surrounding tissues in physiological and pathological conditions. It is postulated that they regulate the formation and integrity of blood vessels. In particular, it has been shown that the Slit/Robo pair plays a novel role in angiogenesis during tumorigenesis and vascular formation during embryogenesis. Herein we summarize briefly the characteristics of this family of neuronal guidance molecules and discuss the extra-neural expression and function of the Slit/Robo pair in angiogenesis in physiological and pathological settings. We report expression of Robo1 protein in capillary endothelium and co-expression of Slit2 and Robo1 proteins in syncytiotrophoblast in healthy term human placental villi. These cellular expression patterns implicate that the Slit/Robo signaling plays an autocrine and/or paracrine role in angiogenesis and trophoblast functions. We also speculate a possible role of this system in pathophysiological placental angiogenesis.

ObjectiveTo elucidate the molecular basis for mitochondrial dysfunction, which has been implicated in the pathogenesis of diabetes complications.Research design and methodsMitochondrial matrix and membrane fractions were generated from liver, brain, heart, and kidney of wild-type and type 1 diabetic Akita mice. Comparative proteomics was performed using label-free proteome expression analysis. Mitochondrial state 3 respirations and ATP synthesis were measured, and mitochondrial morphology was evaluated by electron microscopy. Expression of genes that regulate mitochondrial biogenesis, substrate utilization, and oxidative phosphorylation (OXPHOS) were determined.ResultsIn diabetic mice, fatty acid oxidation (FAO) proteins were less abundant in liver mitochondria, whereas FAO protein content was induced in mitochondria from all other tissues. Kidney mitochondria showed coordinate induction of tricarboxylic acid (TCA) cycle enzymes, whereas TCA cycle proteins were repressed in cardiac mitochondria. Levels of OXPHOS subunits were coordinately increased in liver mitochondria, whereas mitochondria of other tissues were unaffected. Mitochondrial respiration, ATP synthesis, and morphology were unaffected in liver and kidney mitochondria. In contrast, state 3 respirations, ATP synthesis, and mitochondrial cristae density were decreased in cardiac mitochondria and were accompanied by coordinate repression of OXPHOS and peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1alpha transcripts.ConclusionsType 1 diabetes causes tissue-specific remodeling of the mitochondrial proteome. Preservation of mitochondrial function in kidney, brain, and liver, versus mitochondrial dysfunction in the heart, supports a central role for mitochondrial dysfunction in diabetic cardiomyopathy.