Diurnal and circadian regulations by three melatonin receptors in the brain and retina of olive flounder Paralichthys olivaceus: profiles following exogenous melatonin

To establish the molecular basis of circadian rhythm control by melatonin receptors (MTs), we investigated the mitochondrial ribonucleic acid (mRNA) expressions of three types of MTs in different tissues of the olive flounder (Paralichthys olivaceus). All three types of MT mRNAs were expressed in the neural tissues, while MT1 mRNA was expressed in the peripheral tissues and MT2 and MT3 mRNAs were weakly expressed or undetected in these tissues. We observed increased MT mRNA expression in the neural tissues at night under both light–dark (LD) and constant dark (DD) conditions. Although the melatonin-treated cultured pineal gland samples showed similar diurnal variations with high-MT mRNA expression levels at night compared to those of untreated cultured pineal gland samples, the expression levels were considerably higher in the melatonin-treated samples. The plasma melatonin level also significantly increased at night. Under DD conditions, the expression patterns of MT mRNAs were similar to those under the LD photocycle, but the peak was lower and the circadian change patterns were less clear. These findings reinforce the hypothesis that MTs are active in processing light information, and that these genes are regulated by the circadian clock and light, thus suggesting that MTs play an important role in daily and circadian variations in the brain and retina of olive flounders.     

Five new microsatellite loci for Oliver flounder (Paralichthys olivaceus) from an expressed sequence tag (EST) library and cross‐species amplification

Five microsatellite loci have been isolated and characterized from a cDNA library of Oliver flounder, Paralichthys olivaceus. All loci were found to be polymorphic and had between four and 10 alleles. Observed and expected heterozygosities varied from 0.70 to 0.90 and from 0.52 to 0.80, respectively. Five additional fish species assessed for cross‐species amplification revealed between zero and three positive amplifications and between zero and two polymorphic loci per species.     

Sex differences in lifespan extension with acarbose and 17‐α estradiol: gonadal hormones underlie male‐specific improvements in glucose tolerance and mTORC2 signaling

Interventions that extend lifespan in mice can show substantial sexual dimorphism. Here, we show that male‐specific lifespan extension with two pharmacological treatments, acarbose (ACA) and 17‐α estradiol (17aE2), is associated, in males only, with increased insulin sensitivity and improved glucose tolerance. Females, which show either smaller (ACA) or no lifespan extension (17aE2), do not derive these metabolic benefits from drug treatment. We find that these male‐specific metabolic improvements are associated with enhanced hepatic mTORC2 signaling, increased Akt activity, and phosphorylation of FOXO1a – changes that might promote metabolic health and survival in males. By manipulating sex hormone levels through gonadectomy, we show that sex‐specific changes in these metabolic pathways are modulated, in opposite directions, by both male and female gonadal hormones: Castrated males show fewer metabolic responses to drug treatment than intact males, and only those that are also observed in intact females, while ovariectomized females show some responses similar to those seen in intact males. Our results demonstrate that sex‐specific metabolic benefits occur concordantly with sexual dimorphism in lifespan extension. These sex‐specific effects can be influenced by the presence of both male and female gonadal hormones, suggesting that gonadally derived hormones from both sexes may contribute to sexual dimorphism in responses to interventions that extend mouse lifespan.     

Akt‐ and Erk‐mediated regulation of proliferation and differentiation during PDGFRβ‐induced MSC self‐renewal

Understanding the mechanisms that direct mesenchymal stem cell (MSC) self‐renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet‐derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF‐BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl‐2′‐deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT‐PCR, and by long‐term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR‐β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E‐BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ‐induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self‐renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self‐renewal strategies.     

Amyloid‐β‐induced occludin down‐regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation

Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer’s disease (AD). A leaky blood–brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid β (Aβ) peptides of 1–40 and 1–42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Aβ 1–40, the Aβ variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Aβ 1–40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC‐dextran when compared with cells incubated with the scrambled Aβ 1–40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin‐5 and ZO‐1 were unaffected. JNK and p38MAPK inhibition prevented both Aβ 1–40‐mediated down‐regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.     

17β‐Oestradiol promotes differentiation of human embryonic stem cells into dopamine neurons via cross‐talk between insulin‐like growth factors‐1 and oestrogen receptor β

Human embryonic stem cells (hESCs) can self‐renew and differentiate into all cell lineages. E2 is known to exhibit positive effects on embryo development. Although the importance of E2 in many physiological processes has been reported, to date few researchers have investigated the effects of E2 on hESCs differentiation. We studied the effects of E2 on dopamine (DA) neuron induction of hESCs and its related signalling pathways using the three‐stage protocol. In our study, 0.1 μM E2 were applied to hESCs‐derived human embryoid bodies (hEBs) and effects of E2 on neural cells differentiation were investigated. Protein and mRNA level assay indicated that E2 up‐regulated the expression of insulin‐like growth factors (IGF)‐1, ectoderm, neural precursor cells (NPC) and DA neuron markers, respectively. The population of hESC‐derived NPCs and DA neurons was increased to 92% and 93% to that of DMSO group, respectively. Furthermore, yield of DA neuron‐secreted tyrosine hydroxylase (TH) and dopamine was also increased. E2‐caused promotion was relieved in single inhibitor (ICI or JB1) group partly, and E2 effects were repressed more stronger in inhibitors combination (ICI plus JB1) group than in single inhibitor group at hEBs, hNPCs and hDA neurons stages. Owing to oestrogen receptors regulate multiple brain functions, when single or two inhibitors were used to treat neural differentiation stage, we found that oestrogen receptor (ER)β but not ERα is strongly repressed at the hNPCs and hDA neurons stage. These findings, for the first time, demonstrate the molecular cascade and related cell biology events involved in E2‐improved hNPC and hDA neuron differentiation through cross‐talk between IGF‐1 and ERβ in vitro.     

NDV‐D90 inhibits 17β‐estradiol‐mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV‐D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β‐estradiol (E2). We found that NDV‐D90 significantly killed the tumor cell lines MCF‐7 and BT549 in a time‐ and dose‐dependent manner. We also found that NDV‐D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV‐D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF‐7 cells (ER‐positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis‐related proteins through ERα‐and GPER‐independent processes. NDV‐D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER‐negative cells), possibly by impairing E2‐mediated GPER expression. Furthermore, NDV‐D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV‐D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER‐positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.     

Resveratrol protects mice against SEB‐induced acute lung injury and mortality by miR‐193a modulation that targets TGF‐β signalling

Staphylococcal enterotoxin B (SEB) is a potent superantigen produced by Staphylococcus aureus that triggers a strong immune response, characterized by cytokine storm, multi‐organ failure, and often death. When inhaled, SEB can cause acute lung injury (ALI) and respiratory failure. In this study, we investigated the effect of resveratrol (RES), a phytoallexin, on SEB‐driven ALI and mortality in mice. We used a dual‐exposure model of SEB in C3H/HeJ mice, which caused 100% mortality within the first 5 days of exposure, and treatment with RES resulted in 100% survival of these mice up to 10 days post‐SEB exposure. RES reduced the inflammatory cytokines in the serum and lungs, as well as T cell infiltration into the lungs caused by SEB. Treatment with RES also caused increased production of transforming growth factor‐beta (TGF‐β) in the blood and lungs. RES altered the miRNA profile in the immune cells isolated from the lungs. Of these, miR‐193a was strongly induced by SEB and was down‐regulated by RES treatment. Furthermore, transfection studies and pathway analyses revealed that miR‐193a targeted several molecules involved in TGF‐β signalling (TGFβ2, TGFβR3) and activation of apoptotic pathways death receptor‐6 (DR6). Together, our studies suggest that RES can effectively neutralize SEB‐mediated lung injury and mortality through potential regulation of miRNA that promote anti‐inflammatory activities.     

Members of the CIP4 family of proteins participate in the regulation of platelet‐derived growth factor receptor‐β‐dependent actin reorganization and migration

Background information. The F‐BAR {Fes/CIP4 [Cdc42 (cell division cycle 42)‐interacting protein 4] homology and BAR (Bin/amphiphysin/Rvs)} proteins have emerged as important co‐ordinators of signalling pathways that regulate actin assembly and membrane dynamics. The presence of the F‐BAR domain is the hallmark of this family of proteins and the CIP4 (Cdc42‐interacting protein 4) was one of the first identified vertebrate F‐BAR proteins. There are three human CIP4 paralogues, namely CIP4, FBP17 (formin‐binding protein 17) and Toca‐1 (transducer of Cdc42‐dependent actin assembly 1). The CIP4‐like proteins have been implicated in Cdc42‐dependent actin reorganization and in regulation of membrane deformation events visible as tubulation of lipid bilayers. Results. We performed side‐by‐side analyses of the three CIP4 paralogues. We found that the three CIP4‐like proteins vary in their effectiveness to catalyse membrane tubulation and actin reorganization. Moreover, we show that the CIP4‐dependent membrane tubulation is enhanced in the presence of activated Cdc42. Some F‐BAR members have been shown to have a role in the endocytosis of the EGF (epidermal growth factor) receptor and this prompted us to study the involvement of the CIP4‐like proteins in signalling of the PDGFRβ [PDGF (platelet‐derived growth factor) β‐receptor]. We found that knock‐down of CIP4‐like proteins resulted in a prolonged formation of PDGF‐induced dorsal ruffles, as well as an increased PDGF‐dependent cell migration. This was most likely a consequence of a sustained PDGFRβ activation caused by delayed internalization of the receptor in the cells treated with siRNA (small interfering RNA) specific for the CIP4‐like proteins. Conclusions. Our findings show that CIP4‐like proteins induced membrane tubulation downstream of Cdc42 and that they have important roles in PDGF‐dependent actin reorganization and cell migration by regulating internalization and activity of the PDGFRβ. Moreover, the results suggest an important role for the CIP4‐like proteins in the regulation of the activity of the PDGFRβ.     

Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.     

Aβ1–42 oligomer‐induced leakage in an in vitro blood–brain barrier model is associated with up‐regulation of RAGE and metalloproteinases,and down‐regulation of tight junction scaffold proteins

Accumulating evidence indicates that abnormal deposition of amyloid‐β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood–brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ_1–42, and the levels of tight junction (TJ) scaffold proteins (ZO‐1, Claudin‐5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ_1–42, including monomer (Aβ_1–42‐Mono), oligomer (Aβ_1–42‐Oligo), and fibril (Aβ_1–42‐Fibril), our data confirmed that Aβ_1–42‐Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ_1–42 significantly up‐regulated the level of receptor for advanced glycation end‐products (RAGE). Co‐incubation of a polyclonal antibody to RAGE and Aβ_1–42‐Oligo in bEnd.3 cells blocked RAGE suppression of Aβ_1–42‐Oligo‐induced alterations in TJ scaffold proteins and reversed Aβ_1–42‐Oligo‐induced up‐regulation of RAGE, matrix metalloproteinase (MMP)‐2, and MMP‐9. Furthermore, we found that these effects induced by Aβ_1–42‐Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad‐spectrum MMP inhibitor, partially reversed the Aβ_1–42‐Oligo‐induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up‐regulation of MMP‐2 and MMP‐9.

     

β‐sheet breaker peptide inhibitor of Alzheimer's amyloidogenesis with increased blood–brain barrier permeability and resistance to proteolytic degradation in plasma

Short synthetic peptides homologous to the central region of Aβ but bearing proline residues as β‐sheet blockers have been shown in vitro to bind to Aβ with high affinity, partially inhibit Aβ fibrillogenesis, and redissolve preformed fibrils. While short peptides have been used extensively as therapeutic drugs in medicine, two important problems associated with their use in central nervous system diseases have to be addressed: (a) rapid proteolytic degradation in plasma, and (b) poor blood–brain barrier (BBB) permeability. Recently, we have demonstrated that the covalent modification of proteins with the naturally occurring polyamines significantly increases their permeability at the BBB. We have extended this technology to iAβ11, an 11‐residue β‐sheet breaker peptide that inhibits Aβ fibrillogenesis, by covalently modifying this peptide with the polyamine, putrescine (PUT), and evaluating its plasma pharmacokinetics and BBB permeability. After a single intravenous bolus injection in rats, both ¹²⁵I‐YiAβ11 and ¹²⁵I‐PUT‐YiAβ11 showed rapid degradation in plasma as determined by trichloroacetic acid (TCA) precipitation and paper chromatography. By switching to the all d ‐enantiomers of YiAβ11 and PUT‐YiAβ11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively. The permeability coefficient × surface area product at the BBB was five‐ sevenfold higher in the cortex and hippocampus for the ¹²⁵I‐PUT‐d ‐YiAβ11 compared to the ¹²⁵I‐d ‐YiAβ11, with no significant difference in the residual plasma volume. In vitro assays showed that PUT‐d ‐YiAβ11 retains its ability to partially inhibit Aβ fibrillogenesis and dissolve preformed amyloid fibrils. Because of its five‐ to sevenfold increase in permeability at the BBB and its resistance to proteolysis in the plasma, this polyamine‐modified β‐sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vivo and, hence, an important therapy for Alzheimer's disease. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 371–382, 1999