Amphiregulin normalizes altered circuit connectivity for social dominance of the CRTC3 knockout mouse.

Social hierarchy has a profound impact on social behavior, reward processing, and mental health. Moreover, lower social rank can lead to chronic stress and often more serious problems such as bullying victims of abuse, suicide, or attack to society. However, its underlying mechanisms, particularly their association with glial factors, are largely unknown. In this study, we report that astrocyte-derived amphiregulin plays a critical role in the determination of hierarchical ranks. We found that astrocytes-secreted amphiregulin is directly regulated by cAMP response element-binding (CREB)-regulated transcription coactivator 3 (CRTC3) and CREB. Mice with systemic and astrocyte-specific CRTC3 deficiency exhibited a lower social rank with reduced functional connectivity between the prefrontal cortex, a major social hierarchy center, and the parietal cortex. However, this effect was reversed by astrocyte-specific induction of amphiregulin expression, and the epidermal growth factor domain was critical for this action of amphiregulin. These results provide evidence of the involvement of novel glial factors in the regulation of social dominance and may shed light on the clinical application of amphiregulin in the treatment of various psychiatric disorders.

Suppression of Brown Adipocyte Autophagy Improves Energy Metabolism by Regulating Mitochondrial Turnover.

The high abundance of mitochondria and the expression of mitochondrial uncoupling protein 1 (UCP1) confer upon brown adipose tissue (BAT) the unique capacity to convert chemical energy into heat at the expense of ATP synthesis. It was long believed that BAT is present only in infants, and so, it was not considered as a potential therapeutic target for metabolic syndrome; however, the discovery of metabolically active BAT in adult humans has re-stimulated interest in the contributions of BAT metabolic regulation and dysfunction to health and disease. Here we demonstrate that brown adipocyte autophagy plays a critical role in the regulation BAT activity and systemic energy metabolism. Mice deficient in brown adipocyte autophagy due to BAT-specific deletion of Atg7-a gene essential for autophagosome generation-maintained higher mitochondrial content due to suppression of mitochondrial clearance and exhibited improved insulin sensitivity and energy metabolism. Autophagy was upregulated in BAT of older mice compared to younger mice, suggesting its involvement in the age-dependent decline of BAT activity and metabolic rate. These findings suggest that brown adipocyte autophagy plays a crucial role in metabolism and that targeting this pathway may be a potential therapeutic strategy for metabolic syndrome.

Molecular cloning, regulation, and functional analysis of two GHS-R genes in zebrafish.

Mammalian ghrelin is derived from stomach and regulates growth hormone release and appetite by modulating GHS-R (Growth hormone secretagogue receptor) activity. Zebrafish has been developed as a forward genetic screening model system and previous screening identified a number of genes involved in multiple signaling pathways. In this system, ghrelin has been identified and its function and regulation have been shown to be highly conserved to that of mammals. Here, we identified three isoforms of zGHS-R1 and one of zGHS-R2 (zGHS-R2a), and characterized their expression, regulation and function. Three isoforms of zGHS-R1, which we named zGHS-R1a, zGHS-R1b, and zGHS-R1c, are generated by alternative splicing. The expression of zGHS-R1 is highly enriched in brain, intestine tissue, and skin tissues. Compared to zGHS-R1, the expression pattern of zGHS-R2a is rather evenly distributed. A 15 day fasting elevated expression of zGHS-R1 and zGHS-R2 transcripts in anterior intestine tissues, but not in brain. Whereas zGHS-R1a, zGHS-R1c, and zGHS-R2a appear to be presented on the plasma membrane, the localization of zGHS-R1b seems to be restricted in the intracellular region. Treatment of ghrelin agonist, L692,585 or goldfish ghrelin peptides but not rat ghrelin, elevated intracellular Ca(2+) level and phosphorylation of ERK in HEK-293 cells expressing zGHS-R1a, but not zGHS-R1b, zGHS-R1c, or zGHS-R2a. It appears that besides core ghrelin peptide sequence of GS/TSF additional amino acids are required for the activation of zGHS-R1a, as rat ghrelin induces neither intracellular Ca(2+) mobilization nor ERK phosphrylation. These results suggest that ghrelin system in zebrafish is highly conserved to that of mammals, and thus is an ideal in vivo model for dissecting ghrelin system.

Panax red ginseng extract regulates energy expenditures by modulating PKA dependent lipid mobilization in adipose tissue.

Regulation of balance between lipid accumulation and energy consumption is a critical step for the maintenance of energy homeostasis. Here, we show that Panax red ginseng extract treatments increased energy expenditures and prevented mice from diet induced obesity. Panax red ginseng extracts strongly activated Hormone Specific Lipase (HSL) via Protein Kinase A (PKA). Since activation of HSL induces lipolysis in WAT and fatty acid oxidation in brown adipose tissue (BAT), these results suggest that Panax red ginseng extracts reduce HFD induced obesity by regulating lipid mobilization.

Human retinal pigment epithelial cells express the long pentraxin PTX3.

PURPOSE: To determine whether the long pentraxin 3 (PTX3) is expressed in human retinal pigment epithelial cells and is induced by inflammatory cytokines, interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), expression of PTX3 was investigated in the human retinal pigment epithelial cell line, ARPE-19 cells. METHODS: In ARPE-19 cells, we first analyzed PTX3 production in the presence or absence of inflammatory cytokines, IL-1ß, TNF-α, and IFN-γ, dose- and time-dependently using enzyme-linked immunosorbent assay. Protein and mRNA expression of PTX3 was measured with western blotting analysis and real-time reverse transcription-polymerase chain reaction. Specific inhibitors were used to determine the signaling pathways of inflammatory cytokine-induced PTX3 expression. RESULTS: In this study, production of PTX3 was induced by IL-1ß and TNF-α dose- and time-dependently, but not by IFN-γ in ARPE-19 cells. Protein and mRNA expression of PTX3 was significantly upregulated in the presence of IL-1ß and TNF-α. Furthermore, pretreatment with extracellular signal-regulated kinase1/2 and nuclear factor kappa-light-chain-enhancer of activated B cells specific inhibitor abolished IL-1ß and TNF-α-induced PTX3 production, but the other inhibitors had no effect. CONCLUSIONS: These results suggested that human retinal pigment epithelial cells may be a major source of PTX3 production in the presence of proinflammatory cytokines, IL-1ß and TNF-α, and could be an important mediator for host defense and inflammatory response in the retina. The importance of the mitogen-activated protein kinase/extracellular signal-regulated kinase1/2 and nuclear factor kappa-light-chain-enhancer of activated B cells pathways for regulated PTX3 expression may be a potential target for PTX3 regulation in the retina.

Transforming growth factor-ß1 suppression of endotoxin-induced heme oxygenase-1 in macrophages involves activation of Smad2 and downregulation of Ets-2.

Heme oxygenase (HO)-1 is a cytoprotective molecule that is induced during the response to injury. An increase in HO-1 is an acute indicator of inflammation, and early induction of HO-1 has been suggested to correlate with severity of injury. While a great deal is known about the induction of HO-1 by inflammatory mediators and bacterial lipopolysaccharide (LPS), much less is known about the effects of anti-inflammatory mediators on HO-1 expression. Transforming growth factor (TGF)-ß is known to play a critical role in suppressing the immune response, and the TGF-ß1 isoform is expressed in inflammatory cells. Thus, we wanted to investigate whether TGF-ß1 could inhibit the expression of HO-1 during exposure to an inflammatory stimulus in macrophages. Here we demonstrate that TGF-ß1 is able to downregulate LPS-induced HO-1 in mouse macrophages, and this reduction in HO-1 occurred through signaling of TGF-ß1 via its type I receptor, and activation of Smad2. This TGF-ß1 response is dependent on an intact Ets-binding site (EBS) located 93 base pairs upstream from the mouse HO-1 transcription start site. This EBS is known to be important for Ets-2 transactivation of HO-1 by LPS stimulation, and we show that TGF-ß1 is able to suppress LPS-induced Ets-2 mRNA and protein levels in macrophages. Moreover, silencing of Smad2 is able to prevent the suppression of both HO-1 and Ets-2 by TGF-ß1 during exposure to LPS. These data suggest that the return of HO-1 to basal levels during the resolution of an inflammatory response may involve its downregulation by anti-inflammatory mediators.

Nucleotide-binding oligomerization domain protein 2 deficiency enhances neointimal formation in response to vascular injury.

OBJECTIVE: Nucleotide-binding oligomerization domain protein 2 (NOD2) stimulates diverse inflammatory responses resulting in differential cellular phenotypes. To identify the role of NOD2 in vascular arterial obstructive diseases, we investigated the expression and pathophysiological role of NOD2 in a vascular injury model of neointimal hyperplasia. METHODS AND RESULTS: We first analyzed for neointimal hyperplasia following femoral artery injury in NOD2(+/+) and NOD2(-/-) mice. NOD2(-/-) mice showed a 2.86-fold increase in neointimal formation that was mainly composed of smooth muscle (SM) α-actin positive cells. NOD2 was expressed in vascular smooth muscle cells (VSMCs) and NOD2(-/-) VSMCs showed increased cell proliferation in response to mitogenic stimuli, platelet-derived growth factor-BB (PDGF-BB), or fetal bovine serum, compared with NOD2(+/+) VSMCs. Furthermore, NOD2 deficiency markedly promoted VSMCs migration in response to PDGF-BB, and this increased cell migration was attenuated by a phosphatidylinositol 3-kinase inhibitor. However, protein kinase C and c-Jun N-terminal kinase inhibitors exerted negligible effects. Moreover, muramyl dipeptide-stimulated NOD2 prevented PDGF-BB-induced VSMCs migration. CONCLUSION: Functional NOD2 was found to be expressed in VSMCs, and NOD2 deficiency promoted VSMCs proliferation, migration, and neointimal formation after vascular injury. These results provide evidence for the involvement of NOD2 in vascular homeostasis and tissue injury, serving as a potential molecular target in the modulation of arteriosclerotic vascular disease.

CRTC3, a sensor and key regulator for melanogenesis, as a tunable therapeutic target for pigmentary disorders.

Background: Although CREB phosphorylation is known to be essential in UVB/cAMP-stimulated melanogenesis, CREB null mice did not show identifiable pigmentation phenotypes. Here, we show that CREB-regulated transcription co-activator 3 (CRTC3) quantitatively regulates and orchestrates melanogenesis by directly targeting microphthalmia-associated transcription factor (MITF) and regulating the expression of most key melanogenesis-related genes. Methods: We analyzed CRTC3-null, KRT14-SCF transgenic, and their crossover mice. The molecular basis of CRTC3 effects on pigmentation was investigated by histology, melanin/tyrosinase assay, immunoblotting, shRNA, promoter assay, qRT-PCR, and subcellular localization. These analyses were carried out in primary cultured melanocytes, mouse cell lines, normal human cells, co-cultures, and ex vivo human skin. CRTC/CREB activity screening was performed to identify candidate agents for the regulation of melanogenesis. Results: The coat and skin color of CRTC3-null mice was paler due to a reduction in melanin deposition. Melanogenesis-related genes were reduced in CRTC3-deficient cultured melanocytes and tail skin of CRTC3-null mice. Notably, basal levels of MITF present in CRTC3-null mice were sufficient for melanocytic differentiation/survival. Thus CRTC3-null mice showed a comparable number of epidermal melanocytes compared to control mice. Stem cell factor (SCF) introduction by crossing with KRT14-SCF mice increased epidermal melanocytes and melanin deposition in control and CRTC3-null mice, but the skin color remained still light on the CRTC3-null background. Furthermore, we identified the therapeutic potential of altiratinib to inhibit melanogenesis in human melanocytes and human skin effectively and safely. Conclusion: CRTC3 appears to be a key sensor for melanogenesis and can be used as a reversible and tunable tool for selectively regulating melanogenesis without affecting melanocyte integrity. Thus, CRTC3 can also serve as a screening tool for the discovery of ideal melanogenesis-modulating small molecules.

Regulation of heme oxygenase-1 gene by peptidoglycan involves the interaction of Elk-1 and C/EBPalpha to increase expression.

Heme oxygenase (HO)-1 is a cytoprotective enzyme with anti-inflammatory properties. HO-1 is induced during a systemic inflammatory response, and expression of HO-1 is beneficial during sepsis of a Gram-positive source. Systemic infection from Gram-positive organisms has emerged as an important cause of sepsis, with Staphylococcus aureus as a common etiology. An important mediator of Gram-positive infections is peptidoglycan (PGN), a cell wall component of these organisms. Here, we demonstrate that HO-1 played an important, protective role in vivo, as mice deficient in HO-1 were very sensitive to the lethal effects of PGN derived from S. aureus. PGN induced HO-1 protein and mRNA levels, and this regulation occurred at the level of gene transcription. The PGN-responsive region of the HO-1 promoter (from -117 to -66 bp) contains a functional EBS, and Ets proteins are known to be involved in the regulation of inflammatory responses. We showed previously that Ets factors (activators Ets-2 and Ets-1 and repressor Elk-3) regulate HO-1 expression by Gram-negative endotoxin. However, during exposure to a Gram-positive stimulus in the present study, Elk-1 was a potent activator of HO-1 in conjunction with PGN. The ability of Elk-1 to induce HO-1 promoter activity was independent of direct DNA binding, but rather occurred by interacting with the CCAAT/enhancer-binding protein-alpha (C/EBPalpha), which binds to DNA. Moreover, silencing of C/EBPalpha in macrophages prevented induction of HO-1 promoter activity by either Elk-1 or PGN. These data provide further insight into the regulation and function of HO-1 by a mediator of Gram-positive bacteria.

Impaired macrophage autophagy induces systemic insulin resistance in obesity.

Obesity-induced insulin resistance and diabetes are significantly associated with infiltrates of inflammatory cells in adipose tissue. Previous studies recognized the involvement of autophagy in the regulation of metabolism in multiple tissues, including ß-cells, hepatocytes, myocytes, and adipocytes. However, despite the importance of macrophages in obesity-induced insulin resistance, the role of macrophage autophagy in regulating insulin sensitivity is seldom addressed. In the present study, we show that macrophage autophagy is important for the regulation of systemic insulin sensitivity. We found that macrophage autophagy is downregulated by both acute and chronic inflammatory stimuli, and blockade of autophagy significantly increased accumulation of reactive oxygen species (ROS) in macrophages. Macrophage-specific Atg7 knockout mice displayed a shift in the proportion to pro-inflammatory M1 macrophages and impairment of insulin sensitivity and glucose homeostasis under high-fat diet conditions. Furthermore, inhibition of ROS in macrophages with antioxidant recovered adipocyte insulin sensitivity. Our results provide evidence of the underlying mechanism of how macrophage autophagy regulates inflammation and insulin sensitivity. We anticipate our findings will serve as a basis for development of therapeutics for inflammatory diseases, including diabetes.

Platelet-rich fibrin is a Bioscaffold and reservoir of growth factors for tissue regeneration.

The platelet-rich fibrin (PRF) is known as a rich source of autologous cytokines and growth factors and universally used for tissue regeneration in current clinical medicine. However, the microstructure of PRF has not been fully investigated nor have been studied the key molecules that differ PRF from platelet-rich plasma. We fabricated PRF under Choukroun's protocol and produced its extract (PRFe) by freezing at -80°C. The conventional histological, immunohistological staining, and scanning electron microscopy images showed the microstructure of PRF, appearing as two zones, the zone of platelets and the zone of fibrin, which resembled a mesh containing blood cells. The PRFe increased proliferation, migration, and promoted differentiation of the human alveolar bone marrow stem cells (hABMSCs) at 0.5% concentration in vitro. From the results of proteome array, matrix metalloproteinase 9 (MMP9) and Serpin E1 were detected especially in PRFe but not in concentrated platelet-rich plasma. Simultaneous elevation of MMP9, CD44, and transforming growth factor ß-1 receptor was shown at 0.5% PRFe treatment to the hABMSC in immunoblot. Mineralization assay showed that MMP9 directly regulated mineralization differentiation of hABMSC. Transplantation of the fresh PRF into the mouse calvarias enhanced regeneration of the critical-sized defect. Our results strongly support the new characteristics of PRF as a bioscaffold and reservoir of growth factors for tissue regeneration.

Regulating the role of bone morphogenetic protein 4 in tooth bioengineering.

PURPOSE: Culture of the whole organ and regulation of its development using biologic and engineering principles can be used to produce structures and organs for reconstructing defects. The application of these bioengineering approaches in artificial tooth development may be the alternative way to replace missing dentition. MATERIALS AND METHODS: For the artificial bioengineering of a mouse tooth, tooth buds were dissected and transplanted into the diastema of the developing mandible. The mandiblular primordia containing transplanted tooth buds were culture in vitro and in vivo using a bioengineering method. In addition, to regulate the development of tooth germs, bone morphogenetic protein 4 (BMP4) or its antagonist, Noggin was administered. RESULTS: After the period of in vitro and in vivo culture, the transplanted tooth germ in the diastema showed tooth development with supportive structure formation. In the BMP-treated group, the bioengineered tooth was observed with increased maturation of cusp and enamel matrix. However, in the Noggin-treated tooth germs, the developing molar had a crater-like appearance with the immature development of the cusp and suppressed formation of the enamel matrix. CONCLUSIONS: This study confirmed that tooth germ transplantation in the diastema and culture with administration of BMP4 could lead to the mature development of the dental structures. In addition, these results suggest the possibility of bioengineering the tooth in morphogenesis and differentiation even in the toothless area.