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SUMMARYSertoli cells are highly polarized testicular supporting cells that simultaneously nurture progressively maturing germ cells. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, are vital for successful spermatogenesis. However, the mechanisms and developmental timing that underlie the establishment of polarity are poorly understood. To investigate this aspect of testicular function, we conditionally deleted Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Cdc42 deletion disrupted adult Sertoli cell maturation and localization of polarity proteins, but did not affect fetal and early postnatal testicular development, nor the onset of the first wave of spermatogenesis. By early adulthood, however, conditional knockout males exhibited a loss of spermatogenic cells, resulting in a complete lack of sperm. These findings demonstrate that Cdc42 plays an essential role in establishing adult Sertoli cell polarity and, thus, maintaining steady-state spermatogenesis and healthy sperm production.

Supporting cells of the ovary, termed granulosa cells, are essential for ovarian differentiation and oogenesis by providing a nurturing environment for oocyte maintenance and maturation. Granulosa cells are specified in the fetal and perinatal ovary, and sufficient numbers of granulosa cells are critical for the establishment of follicles and the oocyte reserve. Identifying the cellular source from which granulosa cells and their progenitors are derived is an integral part of efforts to understand basic ovarian biology and the etiology of female infertility. In particular, the contribution of mesenchymal cells, especially perivascular cells, to ovarian development is poorly understood but is likely to be a source of new information regarding ovarian function. Here we have identified a cell population in the fetal ovary, which is a Nestin-expressing perivascular cell type. Using lineage tracing and ex vivo organ culture methods, we determined that perivascular cells are multipotent progenitors that contribute to granulosa, thecal, and pericyte cell lineages in the ovary. Maintenance of these progenitors is dependent on ovarian vasculature, likely reliant on endothelial–mesenchymal Notch signaling interactions. Depletion of Nestin + progenitors resulted in a disruption of granulosa cell specification and in an increased number of germ cell cysts that fail to break down, leading to polyovular ovarian follicles. These findings highlight a cell population in the ovary and uncover a key role for vasculature in ovarian differentiation, which may lead to insights into the origins of female gonad dysgenesis and infertility.

Sertoli cells are highly polarized testicular supporting cells that simultaneously nurture multiple stages of germ cells during spermatogenesis. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, is vital for spermatogenesis. However, the mechanisms and developmental timing that underlie Sertoli cell polarity are poorly understood. We investigate this aspect of testicular function by conditionally deleting Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Sertoli Cdc42 deletion leads to increased apoptosis and disrupted polarity of juvenile and adult testes but does not affect fetal and postnatal testicular development. The onset of the first wave of spermatogenesis occurs normally, but it fails to progress past round spermatid stages, and by young adulthood, conditional knockout males exhibit a complete loss of spermatogenic cells. These findings demonstrate that Cdc42 is essential for Sertoli cell polarity and for maintaining steady-state sperm production.

Background Transforming growth factor beta ( TGF ‐β) is an important cytokine in mediating the cardiac fibrosis that often accompanies pathogenic cardiac remodeling. Cardiomyocyte‐specific expression of a mutant αB‐crystallin (Cry AB R 120G ), which causes human desmin‐related cardiomyopathy, results in significant cardiac fibrosis. During onset of fibrosis, fibroblasts are activated to the so‐called myofibroblast state and TGF ‐β binding mediates an essential signaling pathway underlying this process. Here, we test the hypothesis that fibroblast‐based TGF ‐β signaling can result in significant cardiac fibrosis in a disease model of cardiac proteotoxicity that has an exclusive cardiomyocyte‐based etiology. Methods and Results Against the background of cardiomyocyte‐restricted expression of Cry AB R 120G , we have partially ablated TGF ‐β signaling in cardiac myofibroblasts to observe whether cardiac fibrosis is reduced despite the ongoing pathogenic stimulus of Cry AB R 120G production. Transgenic Cry AB R 120G mice were crossed with mice containing a floxed allele of TGF ‐β receptor 2 ( Tgfbr2 f/f ). The double transgenic animals were subsequently crossed to another transgenic line in which Cre expression was driven from the periostin locus ( Postn ) so that Tgfbr2 would be ablated with myofibroblast conversion. Structural and functional assays were then used to determine whether general fibrosis was affected and cardiac function rescued in Cry AB R 120G mice lacking Tgfbr2 in the myofibroblasts. Ablation of myofibroblast specific TGF ‐β signaling led to decreased morbidity in a proteotoxic disease resulting from cardiomyocyte autonomous expression of Cry AB R 120G . Cardiac fibrosis was decreased and hypertrophy was also significantly attenuated, with a significant improvement in survival probability over time, even though the primary proteotoxic insult continued. Conclusions Myofibroblast‐targeted knockdown of Tgfbr2 signaling resulted in reduced fibrosis and improved cardiac function, leading to improved probability of survival.

Introduction: Impairment of protein quality control leads to the accumulation of intracellular misfolded protein aggregates, contributing to cardiac disease and heart failure. Essential protein quality control pathways, including proteasomal degradation and autophagy, are involved in maintaining cellular homeostasis and cardiac function during proteotoxic insult. In a prior study, we undertook an unbiased, total genomewide screen for RNA transcripts and their protein products that affect aggregate accumulations in the cardiomyocytes. Forkhead box F1 (Foxf1) gene was one of the hits from our high throughput screening. In this study, we have performed further validation of Foxf1, a transcription factor and previously unknown player in proteotoxic processes. Aims and Methods: The objective of this study was to validate Foxf1 as a contributor to protein quality control and understand its role in contributing to protein aggregate formation during proteotoxic-induced cardiac disease. We knocked down Foxf1 expression using siRNA mediated transfection in neonatal rat ventricular cardiomyocytes that expressed the protein aggregate inducing mutation of the chaperone α B crystallin (CryABR120G). Proteasomal function and relative activity in cells was analyzed using a degron-destablized green fluorescent protein (GFPu) based reporter protein. Similarly, autophagy activity was determined by autophagic flux assay using bafilomycin treatment. Results: CryABR120G expression leads to aggregate formation and decreased proteasomal function in cardiomyocytes. Knockdown of Foxf1 gene expression in CryABR120G-transfected cardiomyocytes reduced the proteotoxic sequelae, decreasing aggregate concentration. Knockdown of Foxf1 significantly increased proteasomal function in the model without altering autophagy activity. Conclusion: Decreased Foxf1 expression reduced CryABR120G induced aggregate protein accumulation and decreased proteotoxic stress in cardiomyocytes, likely through the maintenance of clearance of the misfolded protein via the proteasomal pathway.

Introduction: Transforming Growth Factor Beta (TGFβ) is an important cytokine in mediating the fibrogenic response and, in particular, cardiac fibrosis. Extensive fibrosis accompanies the cardiac remodeling that occurs during development of the protein conformation-based disease caused by cardiomyocyte-specific expression of a mutant, small, heat shock-like protein and chaperone, aB crystallin (CryABR120G). During the onset of fibrosis, fibroblasts are activated to the so-called “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that TGFβ signaling processes that result in significant cardiac fibrosis in a mouse model of proteotoxic heart disease are mediated by cardiac fibroblasts, rather than cardiomyocytes. Here, we have partially ablated TGFβ signaling only in cardiac myofibroblasts to observe if cardiac fibrosis is reduced. Aims and Methods: The objective of this study was to understand the contributions of fibroblast-derived TGFβ signaling to the development of cardiac fibrosis in a proteotoxic mouse model that results in significant cardiac fibrosis. To test the hypothesis we partially deleted the myofibroblast specific canonical and non-canonical signaling by crossing CryAB R120G mice with Tgfbr1 or Tgfbr2 floxed mice. The double transgene containing mice were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter. Echocardiography, Masson’s Trichome staining, PCR arrays, IHC and western blots were performed to characterize the fibrotic progression in CryAB R120G transgenic mice. Results: We observed that myofibroblast-targeted partial knockdown of Tgf βr1 signaling prolonged survival, modestly reducing fibrosis and improving cardiac function . Similarly, Tgf βr2 partial knockdown prolonged survival, modestly reducing fibrosis without improving cardiac function during fibrosis development in CryAB R120G mice. Conclusion: These findings suggest that, in a model of proteotoxic heart disease, myofibroblast based TGFβ signaling in the heart may contribute to cardiac hypertrophy/dysfunction but cannot account entirely for the fibrotic response.

Cardiac myosin-binding protein C (cMyBP-C) is an integral part of the sarcomeric machinery in cardiac muscle that enables normal function. cMyBP-C regulates normal cardiac contraction by functioning as a brake through interactions with the sarcomere's thick, thin, and titin filaments. cMyBP-C's precise effects as it binds to the different filament systems remain obscure, particularly as it impacts on the myosin heavy chain's head domain, contained within the subfragment 2 (S2) region. This portion of the myosin heavy chain also contains the ATPase activity critical for myosin's function. Mutations in myosin's head, as well as in cMyBP-C, are a frequent cause of familial hypertrophic cardiomyopathy (FHC). We generated transgenic lines in which endogenous cMyBP-C was replaced by protein lacking the residues necessary for binding to S2 (cMyBP-C(S2-)). We found, surprisingly, that cMyBP-C lacking the S2 binding site is incorporated normally into the sarcomere, although systolic function is compromised. We show for the first time the acute and chronic in vivo consequences of ablating a filament-specific interaction of cMyBP-C. This work probes the functional consequences, in the whole animal, of modifying a critical structure-function relationship, the protein's ability to bind to a region of the critical enzyme responsible for muscle contraction, the subfragment 2 domain of the myosin heavy chain. We show that the binding is not critical for the protein's correct insertion into the sarcomere's architecture, but is essential for long-term, normal function in the physiological context of the heart.

Mitochondria, also known as "Power House of cell," are crucial organelles, regulating energy metabolism. Recently, an involvement of mitochondria in cancer occurrence and metastasis has been proposed. The roles of mitochondria in cancer progression/metastasis include alteration of glycolysis, regulation of ROS and suppression of intrinsic apoptosis. This mini-review explains the specific mitochondrial characteristics during cancer metastasis with past and recent findings. It may contribute to understanding mitochondria-related mechanisms of cancer metastasis.

Hyperlipidemia is a major contributor for atherosclerosis and hypolipidemic drugs such as statin are highly prescribed to treat elevated lipid level in plasma. Rubus coreanus, which is widely cultivated in south eastern Asia, have been reported to show significant cholesterol lowering action in hyperlipidemic subjects. Our objective was to determine the cellular effect of Rubus coreanus extract (RCE) on cholesterol biosynthesis in human hepatic cells (HepG2) and to elucidate the molecular mechanism by which it causes change in cholesterol metabolism. RCE treatment lowered cholesterol biosynthesis as well as secretion from HepG2 cells. This effect was associated with lowering the release of apolipoproteins from hepatic cells. RCE treatment also showed an increase in phosphorylation of foxhead box protein 01 (FoXo-1) and 5-adenosine monophosphate-activated protein kinase (AMPK), thus lowering expression of phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase, which might be a major pathway for cholesterol biosynthesis inhibition. Apart from this; RCE also lowered sterol regulatory element-binding protein-1 (SREBP-1) expression in HepG2 cells, showing a long term regulation of cholesterol biosynthesis activity. These results indicate that one of the anti-hyperlipidemic actions of RCE is due to inhibition of cholesterol biosynthesis in hepatic cells and provides first documentation of a hypolipidemic bio-molecular action of Rubus coreanus.

Oxidized low-density lipoprotein (oxLDL) contributes to atherosclerosis in part by being taken up into macrophages via scavenger receptors and leading to foam cell formation. Herbal compounds that have been used to treat blood stasis (a counterpart of atherosclerosis) for centuries include extracts of medicinal plants in the Rosaceae and Leguminosae families. In this study, we investigated the effect of the unripe Rubus coreanus (Korean black raspberry) fruit extract on oxLDL uptake by murine macrophage cells. In the presence of Rubus coreanus extract (RCE), Dil-labeled oxLDL uptake was inhibited in a dose-dependent manner. SP600125, a specific JNK inhibitor, inhibited the uptake of Dil-oxLDL into macrophages. RCE also inhibited JNK phosphorylation in a time- and dose-dependent manner in macrophages treated with oxLDL. These results indicate that among the mitogen-activated protein kinases, JNK phosphorylation is inhibited by RCE, which is likely the mechanism underlying the RCE-induced inhibition of oxLDL uptake by macrophages.

Hyperactivation of phosphoinositol 3-kinase (PI3K) has been suggested to be a potential mechanism for endoplasmic reticulum (ER) stress-enhanced airway hyperresponsiveness, and PI3K inhibitors have been examined as asthma therapeutics. However, the regulatory mechanism linking PI3K to ER stress and related pathological signals in asthma have not been defined. To elucidate these pathogenic pathways, we investigated the influence of a selective PI3Kδ inhibitor, IC87114, on airway inflammation in an ovalbumin/lipopolysaccharide (OVA/LPS)-induced asthma model. In OVA/LPS-induced asthmatic mice, the activity of PI3K, downstream phosphorylation of AKT and activation of nuclear factor-κB (NF-κB) were all significantly elevated; these effects were reversed by IC87114. IC87114 treatment also reduced the OVA/LPS-induced ER stress response by enhancing the intra-ER oxidative folding status through suppression of protein disulfide isomerase activity, ER-associated reactive oxygen species (ROS) accumulation and NOX4 activity. Furthermore, inositol-requiring enzyme-1α (IRE1α)-dependent degradation (RIDD) of IRE1α was reduced by IC87114, resulting in a decreased release of proinflammatory cytokines from bronchial epithelial cells. These results suggest that PI3Kδ may induce severe airway inflammation and hyperresponsiveness by activating NF-κB signaling through ER-associated ROS and RIDD-RIG-I activation. The PI3Kδ inhibitor IC87114 is a potential therapeutic agent against neutrophil-dominant asthma.

Endoplasmic reticulum (ER) stress and associated protein aggregation are closely associated with human diseases, including alterations in hepatic lipid metabolism. Inhibition of ER stress can have a significant effect on the prevention of hepatic dyslipidemia. Here, we studied the role of 4-phenylbutyric acid (4-PBA), a chemical chaperone, on ER stress-induced hepatic lipid accumulation. We studied ER stress induction following CCl4 exposure and delineated mechanisms of the CCl4-induced ER stress response in liver tissue from mice. CCl4 affected the formation of disulfide bonds through excessive hyper-oxidation of protein disulfide isomerase (PDI). Increased complex formation between PDI and its client proteins persisted in CCl4-exposed samples. Conversely, 4-PBA inhibited ER stress via secretion of apolipoprotein B and prevention of hepatic lipid accumulation. We also studied the mechanism-based pharmacokinetic and pharmacodynamic profiles and identified the ER stress-related proteins GRP78 and CHOP, along with plasma apolipoprotein B and triglyceride levels, as novel biomarkers of ER stress-induced hepatic lipid accumulation. ER stress and its clinical relevance for therapeutic approaches were well correlated with the activity of the ER stress regulator 4-PBA, which may be a promising drug candidate for the treatment of hepatic lipid accumulation, such as hepatic steatosis.

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

Cardiovascular science has undergone a revolution in the past 25 years with the application of genetic engineering to the heart and creation of relevant animal models in worms, flies, mice, rats and pigs [1]. A series of evermore-powerful tools have catalyzed the creation of hearts that can express, over-express or not express targeted genes and in some cases, control the developmental timing of the manipulation. Using animal models has allowed the cardiovascular community to establish causality, confirm proof-of-principal for therapeutic approaches, and helped us to understand the relevant pathways and their most important intersections for normal and abnormal cardiac function [1]. The mouse has been the model of choice for the largest number of investigators using these tools as the advantages of dealing with a mammalian, four chambered heart in terms of the data's application to human cardiovascular function and disease remain compelling. The definition of reagents such as the α- and β-myosin heavy chain promoters (MyHC) [2,3] that allowed investigators to drive cardiomyocyte-specific expression at various developmental times and in a chamber-specific manner, cemented the mouse as an important, genetically amenable model for cardiovascular disease. The community's ability to rigorously characterize the resultant phenotype and cardiac physiology using techniques that were first developed for larger animal models, followed quickly [4,5]. The promoters were used to drive expression of a large number of normal and mutated proteins in the heart with the use of the α-MyHC promoter predominating, as it drives cardiomyocyte-specific expression in the atria during fetal development and in all four chambers starting a couple of days before birth, as thyroid hormone production begins and activates transcription from the myc6 locus [3].

Reactive oxygen species exert toxic effects during ischemia-reperfusion (I/R) injury of various organs. This study was designed to evaluate the preventive effects of various isoflavonoids such as biochanin A, daidzein, genistein, rutin and quercetin. These compounds are wellknown naturally occurring compounds with beneficial health effects and antioxidant activity. Free radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and superoxide dismutase (SOD) assay. Among the isoflavonoids tested, biochanine A, quercetin and rutin showed significant DPPH free radical scavenging activity. Similarly, treatment of biochanine A, genistein and rutin significantly increased SOD activity in neonant rat heart myocyte primary cells as well as in H9C2 cells. For ex vivo study, hearts from Sprague-Dawley rats were perfused in Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O(2) and 5% CO(2). Hearts were subjected to 20 min of pre-ischemia followed by 20 min of global ischemia, and then 50 min of reperfusion at 37°C. The test compounds were perfused 10 min before ischemia and during the entire reperfusion period. Among the isoflavonoids tested, only rutin significantly increased left ventricular developed pressure (LVDP) and increased maximum positive and negative dP/dt (+/- dP/dtmax). In left ventricular end diastolic pressure (LVEDP) analysis, rutin, daidzein and biochanin A were effective. Among the isoflavonoids, rutin had consistent protective effects in I/R injury by affecting cardiac dynamic factors as well as by enhancing SOD and DPPH activity.

The role of ER stress on hepatic steatosis was investigated in a rat model. We injected CCl(4) into rats and found that CCl(4) could induce hepatic lipid accumulation, confirmed by Oil Red O staining and by measurement of triglyceride and cholesterol. The expression of ApoB, an apolipoprotein, was decreased in plasma and increased in the liver of CCl(4)-treated animals. The ER stress response was also significantly increased by CCl(4). P450 2E1 expression and activity were increased through interactions of P450 2E1 with NADPH-dependent P450 reductase (NPR) under CCl(4)-treated conditions. In HepG2 cells, intracellular lipid accumulation and its signaling were comparable to in vivo results. In order to elucidate the effect of the ER stress response itself, tunicamycin, an N-acetyl-glycosylation inhibitor, was injected into rats, followed by Oil Red O staining, lipid/triglyceride/cholesterol accumulation analysis, and examination of ApoB expression. Additionally, the ER stress response and upregulation of P450 2E1 were also confirmed in the tunicamycin-treated rats. All of the responses were similar to those seen with CCl(4). The P450 2E1 inhibitor diallyl sulphide (DAS), N-acetylcysteine (NAC), and reduced glutathione (GSH) antioxidants also regulated processes, including ApoB expression and lipid accumulation in CCl(4)-treated animals. In the presence of tunicamycin, DAS or NAC/GSH regulated all of the pathological phenomena with the exception of the ER stress response. In summary, CCl(4) induces liver steatosis, a process involving ER stress-induced P450 2E1 activation and ROS production.

Rubus coreanus fruits have been employed as a traditional medicine for centuries in the Asia-Pacific region. Its pharmacological action differs according to the different extraction methods utilized and the degree of fruit ripening. In this study, we determined the cellular effect of different ethanol extracts of mature and immature Rubus coreanus fruits in human hepatic cell line, HepG2 cells. The antioxidant activity, effect on superoxide dismutase activity and cholesterol biosynthesis efficiency was also evaluated. Immature Rubus coreanus extract showed higher antioxidant capability, compared with that of its mature fractions. Cellular antioxidant proteins including HO-1, Cu/Zn-superoxide dismutase and catalase were highly expressed in the presence of Rubus coreanus. Cholesterol levels in HepG2 cells treated with the water fraction of immature Rubus coreanus were significantly reduced. This antihyperlipidaemic action of Rubus coreanus is a consequence of cholesterol biosynthesis and extracellular secretion in HepG2 cells. These results indicate that among different ethanol fraction of mature and immature Rubus coreanus fruit extracts, water extract of immature fruit extract shows higher antioxidant as well as higher antihyperlipidaemic action.