Starvation-induced transcription factor CREBH negatively governs body growth by controlling GH signaling.

cAMP responsive element-binding protein H (CREBH) is a hepatic transcription factor to be activated during fasting. We generated CREBH knock-in flox mice, and then generated liver-specific CREBH transgenic (CREBH L-Tg) mice in an active form. CREBH L-Tg mice showed a delay in growth in the postnatal stage. Plasma growth hormone (GH) levels were significantly increased in CREBH L-Tg mice, but plasma insulin-like growth factor 1 (IGF1) levels were significantly decreased, indicating GH resistance. In addition, CREBH overexpression significantly increased hepatic mRNA and plasma levels of FGF21, which is thought to be as one of the causes of growth delay. However, the additional ablation of FGF21 in CREBH L-Tg mice could not correct GH resistance at all. CREBH L-Tg mice sustained GH receptor (GHR) reduction and the increase of IGF binding protein 1 (IGFBP1) in the liver regardless of FGF21. As GHR is a first step in GH signaling, the reduction of GHR leads to impairment of GH signaling. These data suggest that CREBH negatively regulates growth in the postnatal growth stage via various pathways as an abundant energy response by antagonizing GH signaling.

Testicular Hypoplasia with Normal Fertility in Neudesin-Knockout Mice.

Neudesin is a secretory protein involved in the brain development during embryonic period and diet-induced development of adipose tissue. Although neudesin is also expressed in the testis, its physiological functions in the testis have not been documented. Therefore, we examined neudesin-encoding neuron-derived neurotrophic factor (Nenf) gene-knockout (Neudesin-KO) mice to clarify the functions of neudesin in the testis. The testicular size of the Neudesin-KO mice was significantly smaller than that of wild-type (WT) mice. However, histological analyses did not reveal any abnormalities in the testis, caput epididymis, and cauda epididymis. Sperm number in the cauda epididymis was comparable between WT and KO mice. Neudesin-KO male mice produced vaginal plugs on paired WT female mice, with a frequency similar to that in WT male mice. A similar number of embryos were developed in the females copulated with WT and Neudesin-KO males. Molecular analysis indicated that the ion transporters Slc19a1 and Kcnk3 were more expressed in the testis of Neudesin-KO mice than in the testis of WT mice, suggesting that the transport of ions and some nutrients in the testis has some abnormalities. Testicular size decreased on postnatal day 6, but not on the day of birth, indicating that neudesin is involved in the postnatal, but not embryonic, development of testis. These results indicate a novel role of neudesin in the development of testis.

The role of increased FGF21 in VLDL-TAG secretion and thermogenic gene expression in mice under protein malnutrition.

Protein malnutrition promotes hepatic lipid accumulation in growing animals. In these animals, fibroblast growth factor 21 (FGF21) rapidly increases in the liver and circulation and plays a protective role in hepatic lipid accumulation. To investigate the mechanism by which FGF21 protects against liver lipid accumulation under protein malnutrition, we determined whether upregulated FGF21 promotes the thermogenesis or secretion of very-low-density lipoprotein (VLDL)-triacylglycerol (TAG). The results showed that protein malnutrition decreased VLDL-TAG secretion, but the upregulation of FGF21 did not oppose this effect. In addition, protein malnutrition increased expression of the thermogenic gene uncoupling protein 1 in inguinal white adipose and brown adipose tissue in an FGF21-dependent manner. However, surgically removing inguinal white adipose tissue did not affect liver triglyceride levels in protein-malnourished mice. These data suggest that FGF21 stimulates thermogenesis under protein malnutrition, but this is not the causative factor underlying the protective role of FGF21 against liver lipid accumulation.

The hepatokine FGF21 is crucial for peroxisome proliferator-activated receptor-α agonist-induced amelioration of metabolic disorders in obese mice.

Obesity causes excess fat accumulation in white adipose tissues (WAT) and also in other insulin-responsive organs such as the skeletal muscle, increasing the risk for insulin resistance, which can lead to obesity-related metabolic disorders. Peroxisome proliferator-activated receptor-α (PPARα) is a master regulator of fatty acid oxidation whose activator is known to improve hyperlipidemia. However, the molecular mechanisms underlying PPARα activator-mediated reduction in adiposity and improvement of metabolic disorders are largely unknown. In this study we investigated the effects of PPARα agonist (fenofibrate) on glucose metabolism dysfunction in obese mice. Fenofibrate treatment reduced adiposity and attenuated obesity-induced dysfunctions of glucose metabolism in obese mice fed a high-fat diet. However, fenofibrate treatment did not improve glucose metabolism in lipodystrophic A-Zip/F1 mice, suggesting that adipose tissue is important for the fenofibrate-mediated amelioration of glucose metabolism, although skeletal muscle actions could not be completely excluded. Moreover, we investigated the role of the hepatokine fibroblast growth factor 21 (FGF21), which regulates energy metabolism in adipose tissue. In WAT of WT mice, but not of FGF21-deficient mice, fenofibrate enhanced the expression of genes related to brown adipocyte functions, such as Ucp1, Pgc1a, and Cpt1b Fenofibrate increased energy expenditure and attenuated obesity, whole body insulin resistance, and adipocyte dysfunctions in WAT in high-fat-diet-fed WT mice but not in FGF21-deficient mice. These findings indicate that FGF21 is crucial for the fenofibrate-mediated improvement of whole body glucose metabolism in obese mice via the amelioration of WAT dysfunctions.

Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress.

AIMS/HYPOTHESIS: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that exhibits anti-diabetic and anti-obesity activity. FGF21 expression is increased in patients with and mouse models of obesity or nonalcoholic fatty liver disease (NAFLD). However, the functional role and molecular mechanism of FGF21 induction in obesity or NAFLD are not clear. As endoplasmic reticulum (ER) stress is triggered in obesity and NAFLD, we investigated whether ER stress affects FGF21 expression or whether FGF21 induction acts as a mechanism of the unfolded protein response (UPR) adaptation to ER stress induced by chemical stressors or obesity. METHODS: Hepatocytes or mouse embryonic fibroblasts deficient in UPR signalling pathways and liver-specific eIF2α mutant mice were employed to investigate the in vitro and in vivo effects of ER stress on FGF21 expression, respectively. The in vivo importance of FGF21 induction by ER stress and obesity was determined using inducible Fgf21-transgenic mice and Fgf21-null mice with or without leptin deficiency. RESULTS: We found that ER stressors induced FGF21 expression, which was dependent on a PKR-like ER kinase-eukaryotic translation factor 2α-activating transcription factor 4 pathway both in vitro and in vivo. Fgf21-null mice exhibited increased expression of ER stress marker genes and augmented hepatic lipid accumulation after tunicamycin treatment. However, these changes were attenuated in inducible Fgf21-transgenic mice. We also observed that Fgf21-null mice with leptin deficiency displayed increased hepatic ER stress response and liver injury, accompanied by deteriorated metabolic variables. CONCLUSIONS/INTERPRETATION: Our results suggest that FGF21 plays an important role in the adaptive response to ER stress- or obesity-induced hepatic metabolic stress.

Rapid increase in fibroblast growth factor 21 in protein malnutrition and its impact on growth and lipid metabolism.

Protein malnutrition promotes hepatic steatosis, decreases insulin-like growth factor (IGF)-I production and retards growth. To identify new molecules involved in such changes, we conducted DNA microarray analysis on liver samples from rats fed an isoenergetic low-protein diet for 8 h. We identified the fibroblast growth factor 21 gene (Fgf21) as one of the most strongly up-regulated genes under conditions of acute protein malnutrition (P<0·05, false-discovery rate<0·001). In addition, amino acid deprivation increased Fgf21 mRNA levels in rat liver-derived RL-34 cells (P<0·01). These results suggested that amino acid limitation directly increases Fgf21 expression. FGF21 is a polypeptide hormone that regulates glucose and lipid metabolism. FGF21 also promotes a growth hormone-resistance state and suppresses IGF-I in transgenic mice. Therefore, to determine further whether Fgf21 up-regulation causes hepatic steatosis and growth retardation after IGF-I decrease in protein malnutrition, we fed an isoenergetic low-protein diet to Fgf21-knockout (KO) mice. Fgf21-KO did not rescue growth retardation and reduced plasma IGF-I concentration in these mice. Fgf21-KO mice showed greater epididymal white adipose tissue weight and increased hepatic TAG and cholesterol levels under protein malnutrition conditions (P<0·05). Overall, the results showed that protein deprivation directly increased Fgf21 expression. However, growth retardation and decreased IGF-I were not mediated by increased FGF21 expression in protein malnutrition. Furthermore, FGF21 up-regulation rather appears to have a protective effect against obesity and hepatic steatosis in protein-malnourished animals.

Expression of Fgf23 in activated dendritic cells and macrophages in response to immunological stimuli in mice.

Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse biological activities. While several studies have revealed that Fgf23 plays important roles in the regulation of phosphate and vitamin D metabolism, the additional physiological roles of Fgf23 remain unclear. Although it is believed that osteoblasts/osteocytes are the main sources of Fgf23, we previously found that Fgf23 mRNA is also expressed in the mouse thymus, suggesting that it might be involved in the immune system. In this study we examined the potential roles of Fgf23 in immunological responses. Mouse serum Fgf23 levels were significantly increased following inoculation with Escherichia coli or Staphylococcus aureus or intraperitoneal injection of lipopolysaccharide. We also identified activated dendritic cells and macrophages that potentially contributed to increased serum Fgf23 levels. Nuclear factor-kappa B (NF-κB) signaling was essential for the induction of Fgf23 expression in dendritic cells in response to immunological stimuli. Moreover, we examined the effects of recombinant Fgf23 protein on immune cells in vitro. Fgfr1c, a potential receptor for Fgf23, was abundantly expressed in macrophages, suggesting that Fgf23 might be involved in signal transduction in these cells. Our data suggest that Fgf23 potentially increases the number in macrophages and induces expression of tumor necrosis factor-α (TNF-α), a proinflammatory cytokine. Collectively, these data suggest that Fgf23 might be intimately involved in inflammatory processes.

Angiotensin II-induced cardiac hypertrophy and fibrosis are promoted in mice lacking Fgf16.

Fibroblast growth factors (Fgfs) are pleiotropic proteins involved in development, repair and metabolism. Fgf16 is predominantly expressed in the heart. However, as the heart function is essentially normal in Fgf16 knockout mice, its role has remained unclear. To elucidate the pathophysiological role of Fgf16 in the heart, we examined angiotensin II-induced cardiac hypertrophy and fibrosis in Fgf16 knockout mice. Angiotensin II-induced cardiac hypertrophy and fibrosis were significantly promoted by enhancing Tgf-ß1 expression in Fgf16 knockout mice. Unexpectedly, the response to cardiac remodeling was apparently opposite to that in Fgf2 knockout mice. These results indicate that Fgf16 probably prevents cardiac remodeling, although Fgf2 promotes it. Cardiac Fgf16 expression was induced after the induction of Fgf2 expression by angiotensin II. In cultured cardiomyocytes, Fgf16 expression was promoted by Fgf2. In addition, Fgf16 antagonized Fgf2-induced Tgf-ß1 expression in cultured cardiomyocytes and noncardiomyocytes. These results suggest a possible mechanism whereby Fgf16 prevents angiotensin II-induced cardiac hypertrophy and fibrosis by antagonizing Fgf2. The present findings should provide new insights into the roles of Fgf signaling in cardiac remodeling.

A secretory protein neudesin regulates splenic red pulp macrophages in erythrophagocytosis and iron recycling.

Neudesin, originally identified as a neurotrophic factor, has primarily been studied for its neural functions despite its widespread expression. Using 8-week-old neudesin knockout mice, we elucidated the role of neudesin in the spleen. The absence of neudesin caused mild splenomegaly, shortened lifespan of circulating erythrocytes, and abnormal recovery from phenylhydrazine-induced acute anemia. Blood cross-transfusion and splenectomy experiments revealed that the shortened lifespan of erythrocytes was attributable to splenic impairment. Further analysis revealed increased erythrophagocytosis and decreased iron stores in the splenic red pulp, which was linked to the upregulation of Fcγ receptors and iron-recycling genes in neudesin-deficient macrophages. In vitro analysis confirmed that neudesin suppressed erythrophagocytosis and expression of Fcγ receptors through ERK1/2 activation in heme-stimulated macrophages. Finally, we observed that 24-week-old neudesin knockout mice exhibited severe symptoms of anemia. Collectively, our results suggest that neudesin regulates the function of red pulp macrophages and contributes to erythrocyte and iron homeostasis.

BACH1 inhibits senescence, obesity, and short lifespan by ferroptotic FGF21 secretion.

Ferroptosis is a type of regulated cell death characterized by iron-dependent lipid peroxidation. A model cell system is constructed to induce ferroptosis by re-expressing the transcription factor BACH1, a potent ferroptosis inducer, in immortalized mouse embryonic fibroblasts (iMEFs). The transfer of the culture supernatant from ferroptotic iMEFs activates the proliferation of hepatoma cells and other fibroblasts and suppresses cellular senescence-like features. The BACH1-dependent secretion of the longevity factor FGF21 is increased in ferroptotic iMEFs. The anti-senescent effects of the culture supernatant from these iMEFs are abrogated by Fgf21 knockout. BACH1 activates the transcription of Fgf21 by promoting ferroptotic stress and increases FGF21 protein expression by suppressing its autophagic degradation through transcriptional Sqstm1 and Lamp2 repression. The BACH1-induced ferroptotic FGF21 secretion suppresses obesity in high-fat diet-fed mice and the short lifespan of progeria mice. The inhibition of these aging-related phenotypes can be physiologically significant regarding ferroptosis.

Sostdc1 defines the size and number of skin appendage placodes.

Mammary glands and hair follicles develop as ectodermal organs sharing common features during embryonic morphogenesis. The molecular signals controlling the initiation and patterning of skin appendages involve the bone morphogenetic proteins and Wnt family members, which are commonly thought to serve as inhibitory and activating cues, respectively. Here, we have examined the role of the Bmp and Wnt pathway modulator Sostdc1 in mammary gland, and hair and vibrissa follicle development using Sostdc1-null mice. Contrary to previous speculations, loss of Sostdc1 did not affect pelage hair cycling. Instead, we found that Sostdc1 limits the number of developing vibrissae and other muzzle hair follicles, and the size of primary hair placodes. Sostdc1 controls also the size and shape of mammary buds. Furthermore, Sostdc1 is essential for suppression of hair follicle fate in the normally hairless nipple epidermis, but its loss also promotes the appearance of supernumerary nipple-like protrusions. Our data suggest that functions of Sostdc1 can be largely attributed to its ability to attenuate Wnt/ß-catenin signaling.

A putative polypeptide N-acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis.

We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294-3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.

Growth hormone induces hepatic production of fibroblast growth factor 21 through a mechanism dependent on lipolysis in adipocytes.

Fibroblast growth factor (FGF) 21 and growth hormone (GH) are metabolic hormones that play important roles in regulating glucose and lipid metabolism. Both hormones are induced in response to fasting and exert their actions on adipocytes to regulate lipolysis. However, the molecular interaction between these two hormones remains unclear. Here we demonstrate the existence of a feedback loop between GH and FGF21 on the regulation of lipolysis in adipocytes. A single bolus injection of GH into C57 mice acutely increases both mRNA and protein expression of FGF21 in the liver, thereby leading to a marked elevation of serum FGF21 concentrations. Such a stimulatory effect of GH on hepatic FGF21 production is abrogated by pretreatment of mice with the lipolysis inhibitor niacin. Direct incubation of either liver explants or human HepG2 hepatocytes with GH has no effect on FGF21 expression. On the other hand, FGF21 production in HepG2 cells is significantly induced by incubation with the conditioned medium harvested from GH-treated adipose tissue explants, which contains high concentrations of free fatty acids (FFA). Further analysis shows that FFA released by GH-induced lipolysis stimulates hepatic FGF21 expression by activation of the transcription factor PPARα. In FGF21-null mice, both the magnitude and duration of GH-induced lipolysis are significantly higher than those in their wild type littermates. Taken together, these findings suggest that GH-induced hepatic FGF21 production is mediated by FFA released from adipose tissues, and elevated FGF21 in turn acts as a negative feedback signal to terminate GH-stimulated lipolysis in adipocytes.

New developments in the biology of fibroblast growth factors.

The fibroblast growth factor (FGF) family is composed of 18 secreted signaling proteins consisting of canonical FGFs and endocrine FGFs that activate four receptor tyrosine kinases (FGFRs 1-4) and four intracellular proteins (intracellular FGFs or iFGFs) that primarily function to regulate the activity of voltage-gated sodium channels and other molecules. The canonical FGFs, endocrine FGFs, and iFGFs have been reviewed extensively by us and others. In this review, we briefly summarize past reviews and then focus on new developments in the FGF field since our last review in 2015. Some of the highlights in the past 6 years include the use of optogenetic tools, viral vectors, and inducible transgenes to experimentally modulate FGF signaling, the clinical use of small molecule FGFR inhibitors, an expanded understanding of endocrine FGF signaling, functions for FGF signaling in stem cell pluripotency and differentiation, roles for FGF signaling in tissue homeostasis and regeneration, a continuing elaboration of mechanisms of FGF signaling in development, and an expanding appreciation of roles for FGF signaling in neuropsychiatric diseases. This article is categorized under: Cardiovascular Diseases > Molecular and Cellular Physiology Neurological Diseases > Molecular and Cellular Physiology Congenital Diseases > Stem Cells and Development Cancer > Stem Cells and Development.

Social stress alters sleep in FGF21-deficient mice.

Although several previous studies have suggested a relationship between sleep and the stress response, the mechanism underlying this relationship remains largely unknown. Here, we show that fibroblast growth factor 21 (FGF21), a lipid metabolism-related hormone, may play a role in this relationship. In this study, we examined differences in the stress response between FGF21 knockout (KO) mice and wild-type (WT) mice after social defeat stress (SDS). When the amount of non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep and wakefulness were averaged over the dark period after SDS, only KO mice showed significant differences in NREM sleep and wakefulness. In the social interaction test, KO mice seemed to be more prone to social avoidance. Our real-time (RT) -PCR results revealed that the mRNA expression of the stress- and sleep-related gene gamma-aminobutyric acid A receptor subunit alpha 2 was significantly lower in WT mice than in KO mice. Moreover, KO mice showed lower plasma levels of ketone bodies, which also affect sleep/wake regulation, than WT mice. These results suggested that FGF21 might influence sleep/wake regulation by inducing production of an anti-stress agent and/or ketone bodies, which may result in resilience to social stress.

Time-imposed daily restricted feeding induces rhythmic expression of Fgf21 in white adipose tissue of mice.

Fibroblast growth factor 21 (FGF21) is a key metabolic regulator that is induced by fasting and starvation, and its expression is thought to be regulated by the circadian clock in the liver. To evaluate the functional role of FGF21 in the circadian regulation of physiology and behavior, we examined the temporal expression profiles of Fgf21 and circadian clock genes in addition to behavioral activity rhythms under adlibitum feeding (ALF) and time-imposed restricted feeding (RF) in mice. Four hours of daily restricted feeding during the daytime induced over an 80-fold increase in feeding-dependent rhythmic Fgf21 mRNA expression in epididymal white adipose tissue (eWAT), although the expression levels were continuously increased 10-fold in the liver of wild-type (WT) mice. Refeeding subsequent to transient fasting revealed that refeeding but not fasting remarkably induces Fgf21 expression in eWAT, although fasting-induced hepatic Fgf21 expression is completely reversed by refeeding. The free-running period of locomotor activity rhythm under ALF and the food anticipatory activity (FAA) under RF remained intact in Fgf21 knockout (KO) mice, suggesting that FGF21 is dispensable for both the central clock in the suprachiasmatic nucleus (SCN) and the food-entrainable oscillator that governs the FAA. Temporal expression profiles of circadian genes such as mPer2 and BMAL1 were essentially identical in both tissues between WT and Fgf21 KO mice under RF. The physiological role of the refeeding-induced adipose Fgf21 expression remains to be elucidated.

Fgf20b is required for the ectomesenchymal fate establishment of cranial neural crest cells in zebrafish.

In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish.

Enterohepatic Transcription Factor CREB3L3 Protects Atherosclerosis via SREBP Competitive Inhibition.

BACKGROUND & AIMS: cAMP responsive element-binding protein 3 like 3 (CREB3L3) is a membrane-bound transcription factor involved in the maintenance of lipid metabolism in the liver and small intestine. CREB3L3 controls hepatic triglyceride and glucose metabolism by activating plasma fibroblast growth factor 21 (FGF21) and lipoprotein lipase. In this study, we intended to clarify its effect on atherosclerosis. METHODS: CREB3L3-deficifient, liver-specific CREB3L3 knockout, intestine-specific CREB3L3 knockout, both liver- and intestine-specific CREB3L3 knockout, and liver CREB3L3 transgenic mice were crossed with LDLR-/- mice. These mice were fed with a Western diet to develop atherosclerosis. RESULTS: CREB3L3 ablation in LDLR-/- mice exacerbated hyperlipidemia with accumulation of remnant APOB-containing lipoprotein. This led to the development of enhanced aortic atheroma formation, the extent of which was additive between liver- and intestine-specific deletion. Conversely, hepatic nuclear CREB3L3 overexpression markedly suppressed atherosclerosis with amelioration of hyperlipidemia. CREB3L3 directly up-regulates anti-atherogenic FGF21 and APOA4. In contrast, it antagonizes hepatic sterol regulatory element-binding protein (SREBP)-mediated lipogenic and cholesterogenic genes and regulates intestinal liver X receptor-regulated genes involved in the transport of cholesterol. CREB3L3 deficiency results in the accumulation of nuclear SREBP proteins. Because both transcriptional factors share the cleavage system for nuclear transactivation, full-length CREB3L3 and SREBPs in the endoplasmic reticulum (ER) functionally inhibit each other. CREB3L3 promotes the formation of the SREBP-insulin induced gene 1 complex to suppress SREBPs for ER-Golgi transport, resulting in ER retention and inhibition of proteolytic activation at the Golgi and vice versa. CONCLUSIONS: CREB3L3 has multi-potent protective effects against atherosclerosis owing to new mechanistic interaction between CREB3L3 and SREBPs under atherogenic conditions.

Fgf4 is required for left-right patterning of visceral organs in zebrafish.

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffer's vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffer's vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffer's vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.

Neuferricin, a novel extracellular heme-binding protein, promotes neurogenesis.

We identified a novel extracellular heme-binding protein and named it neuferricin. The recombinant mouse neuferricin produced in High Five cells was secreted efficiently into the culture medium. Mouse neuferricin mRNA was expressed mainly in the brain at the embryo stage and gradually increased during development. At postnatal stage, it was widely expressed in the brain, heart, adrenal gland, and kidney. Mouse neuferricin has 263 amino acids. It has a cytochrome b5-like heme/steroid-binding domain and appeared to bind hemin because neuferricin solution, but not a solution of neuferricinDeltaHBD (a mutant lacking the heme-binding domain), was tinged with brown and had an absorbance peak at 402 nm. In addition, the experiment with anti-neuferricin antibody using heme-affinity chromatography proved that the endogenous neuferricin detected in the culture medium of Neuro2a cells was associated with hemin. Inhibition of endogenous neuferricin by RNA interference excessively promoted cell survival and proliferation and suppressed neurite outgrowth during the induction of differentiation in Neuro2a cells. Addition of recombinant mouse neuferricin, but not neuferricinDeltaHBD, suppressed survival of Neuro2a cells and rescued from the effects of neuferricin RNAi. In primary cultured mouse neural precursor cells, recombinant mouse neuferricin exhibited the ability to promote neurogenesis. The identification of neuferricin, a novel extracellular heme-binding protein with cytochrome b5-like heme/steroid-binding domain and its neurogenic activity, provide new insights not only into brain development but also the function of heme-binding proteins as extracellular signal transmitters.