STK25 and MST3 Have Overlapping Roles to Regulate Rho GTPases during Cortical Development.

Precise control of neuronal migration is required for the laminar organization of the neocortex and critical for brain function. We previously reported that the acute disruption of the Stk25 gene (Stk25 conditional knock-out; cKO) during mouse embryogenesis causes anomalous neuronal migration in the neocortex, but paradoxically the Stk25 cKO did not have a cortical phenotype, suggesting some forms of compensation exist. In this study, we report that MST3, another member of the GCKIII subgroup of the Ste20-like kinase family, compensates for loss of Stk25 and vice versa with sex independent manner. MST3 overexpression rescued neuronal migration deficit and abnormal axonogenesis in Stk25 cKO brains. Mechanistically, STK25 leads to Rac1 activation and reduced RhoA levels in the developing brain, both of which are required to fully restore neuronal migration in the Stk25 cKO brain. Abnormal migration phenotypes are also rescued by overexpression of Bacurd1and Cul3, which target RhoA for degradation, and activate Rac1. This study reveals that MST3 upregulation is capable of rescuing acute Stk25 deficiency and resolves details of signaling downstream STK25 required for corticogenesis both common to and distinct from MST3 signaling.SIGNIFICANCE STATEMENT Proper neuronal migration during cortical development is required for normal neuronal function. Here, we show that STK25 and MST3 kinases regulate neuronal migration and polarization in a mutually compensatory manner. Furthermore, STK25 balances Rac1 activity and RhoA level through forming complexes with α-PIX and ß-PIX, GTPase regulatory enzymes, and Cullin3-Bacurd1/Kctd13, a pair of RhoA ubiquitination molecules in a kinase activity-independent manner. Our findings demonstrate the importance of overlapping and unique roles of STK25 and MST3 to regulate Rho GTPase activities in cortical development.

DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.

In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells.

Unveiling the vitamin E profile in rice bran extracellular vesicles: evaluation of extraction and preparation methods.

Extracellular vesicles (EVs) are nanoscale entities secreted by various cells, encapsulating various nucleic acids and proteins that play important roles in cellular activities. Although rice bran is known for its richness in phytochemicals such as tocopherol and tocotrienol, the distribution of these compounds within EVs has not been extensively studied. The objective of this study was to detect and analyze the presence of vitamin E in EVs extracted from rice bran. We investigated several EV extraction methods, including rotation, vortex mixing, and ultrasonication, followed by post-extraction techniques such as ultracentrifugation, ultrafiltration, and lyophilization. Vitamin E in the EVs from rice bran was analyzed using LC-FLD. This study is the first to identify tocopherol and tocotrienol in rice bran-derived EVs. Our results indicate that ultracentrifugation followed by rotation is the most effective method for the preparation of rice bran-derived EVs. Notably, the vitamin E profile in EVs varies depending on the preparation method and differs from that in rice bran extracts. The pronounced presence of vitamin E in EVs suggests unique pharmacokinetics and underscores the potential of EVs as carriers for drug delivery systems. This study not only confirms the presence of vitamin E in EVs, but also underscores the potential of EVs and their phytochemical content for therapeutic applications.

The distribution of neuroligin4, an autism-related postsynaptic molecule, in the human brain.

NLGN4X was identified as a single causative gene of rare familial nonsyndromic autism for the first time. It encodes the postsynaptic membrane protein Neuroligin4 (NLGN4), the functions and roles of which, however, are not fully understood due to the lack of a closely homologous gene in rodents. It has been confirmed only recently that human NLGN4 is abundantly expressed in the cerebral cortex and is localized mainly to excitatory synapses. However, the detailed histological distribution of NLGN4, which may have important implications regarding the relationships between NLGN4 and autistic phenotypes, has not been clarified. In this study, we raised specific monoclonal and polyclonal antibodies against NLGN4 and examined the distribution of NLGN4 in developing and developed human brains by immunohistochemistry. We found that, in the brain, NLGN4 is expressed almost exclusively in neurons, in which it has a widespread cytoplasmic pattern of distribution. Among various types of neurons with NLGN4 expression, we identified consistently high expression of NLGN4 in hypothalamic oxytocin (OXT)/vasopressin (AVP)-producing cells. Quantitative analyses revealed that the majority of OXT/AVP-producing neurons expressed NLGN4. NLGN4 signals in other large neurons, such as pyramidal cells in the cerebral cortex and hippocampus as well as neurons in the locus coeruleus and the raphe nucleus, were also remarkable, clearly contrasting with no or scarce signals in Purkinje cells. These data suggest that NLGN4 functions in systems involved in intellectual abilities, social abilities, and sleep and wakefulness, impairments of which are commonly seen in autism.

Hesperetin upregulates ABCA1 expression and promotes cholesterol efflux from THP-1 macrophages.

ABCA1, a member of the ATP-binding cassette transporter family, regulates high-density lipoprotein (HDL) metabolism and cholesterol transport. Its expression is upregulated mainly by the activation of the liver X receptor (LXR). Since ABCA1 plays a pivotal role in cholesterol and HDL metabolism, identification of a compound capable of increasing its expression may be beneficial for the prevention and therapy of atherosclerosis. Firefly luciferase reporter assays were developed for human ABCA1 promoters and LXR enhancers, and an in-house phytochemical library was screened. It was found that a citrus flavonoid, hesperetin (1), increased ABCA1 promoter and LXR enhancer activities in THP-1 macrophages. It was also found that this flavonoid promoted PPAR-enhancing activity. In accordance with these findings, 1 increased mRNA and protein expression of ABCA1 and consequently upregulated ApoA-I-mediated cholesterol efflux. These results provide evidence that 1 promotes ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression through the activation of LXRα and PPARγ.

Microvesicle-mediated RNA molecule delivery system using monocytes/macrophages.

Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, are possible vehicles for navigating RNA molecules to body tissues. It is considered that intravenous injection of such MVs or exosomes from patients would not cause severe not-self and toxic reactions. Previously, we found that macrophages take up liposome-entrapped RNA molecules, some of which remain undegraded in the cells. Here, we demonstrate that transfected RNA molecules in human monocytic leukemia THP-1 cells were shed from THP-1 macrophages as contents in MVs during incubation in serum-free medium, which shedding was shown by biochemical analyses such as quantitative reverse transcription (qRT)-PCR, expression of TSG101 (a membrane-associated exosomal protein), and immunoelectron microscopic study. More chemically modified RNA molecules (miR-143BPs) entrapped by MVs (MV-miR-143BPs) were secreted from THP-1 macrophages after miR-143BP transfection compared with the amount after transfection with nonmodified miR-143 transfection. Furthermore, we show that the THP-1 macrophages, which were transfected with the miR-143BP ex vivo, secreted MV-miR-143BPs in xenografted nude mice after intravenous injection, because miR-143 levels were significantly increased in the serum, tumor, and kidney of the host animals. These data suggest that some of the transfected miR-143BPs were secreted from THP-1 macrophages as MV-RNAs both in vitro and in vivo.

Identification of non-coding RNAs embracing microRNA-143/145 cluster.

In a variety of cancers, altered patterns of microRNA (miRNA) expression are reported and may affect the cell cycle and cell survival. Recent studies suggest that the expression level of miRNAs that act as tumor suppressors is frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription and disturbances in miRNA processing. miR-143 and -145, which are located approximately 1.3 kb from each other at chromosome 5q33, are highly expressed in several tissues, but down-regulated in most cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that both miRNAs were expressed well under the same control program in human tissues, but were down-regulated equally in the most of the cancer cell lines tested. Then we identified the host gene encoding both miRNAs. The transcripts of this gene were approximately 11, 7.5, and 5.5 kb long; and the expression of these transcripts was coordinated with that of its resident miRNAs and down-regulated in the cancer cell lines tested as well as in colorectal cancer tissue samples. These data demonstrate that the host gene can function as a primary miRNA transcript and suggest that the down-regulation of host gene expression caused the low-expression of its encoded microRNAs-143 and -145 in human cancer cell lines and in cancer tissues.

Knockdown of Son, a mouse homologue of the ZTTK syndrome gene, causes neuronal migration defects and dendritic spine abnormalities.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, a rare congenital anomaly syndrome characterized by intellectual disability, brain malformation, facial dysmorphism, musculoskeletal abnormalities, and some visceral malformations is caused by de novo heterozygous mutations of the SON gene. The nuclear protein SON is involved in gene transcription and RNA splicing; however, the roles of SON in neural development remain undetermined. We investigated the effects of Son knockdown on neural development in mice and found that Son knockdown in neural progenitors resulted in defective migration during corticogenesis and reduced spine density on mature cortical neurons. The induction of human wild-type SON expression rescued these neural abnormalities, confirming that the abnormalities were caused by SON insufficiency. We also applied truncated SON proteins encoded by disease-associated mutant SON genes for rescue experiments and found that a truncated SON protein encoded by the most prevalent SON mutant found in ZTTK syndrome rescued the neural abnormalities while another much shorter mutant SON protein did not. These data indicate that SON insufficiency causes neuronal migration defects and dendritic spine abnormalities, which seem neuropathological bases of the neural symptoms of ZTTK syndrome. In addition, the results support that the neural abnormalities in ZTTK syndrome are caused by SON haploinsufficiency independent of the types of mutation that results in functional or dysfunctional proteins.

Decreased expression of microRNA-143 and -145 in human gastric cancers.

OBJECTIVE: Downregulation of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. We investigated the role of the miRNAs miR-143 and miR-145 in gastric cancers. METHODS: The expression levels of miR-143 and miR-145 in the samples from 43 patients with gastric cancer were determined by real-time PCR using TaqMan assay. The growth inhibitory effect was estimated by the transfection of human gastric cancer cells with the miRNA. RESULTS: The expression levels of miR-143 and -145 were decreased in most human gastric cancers examined, as previously reported to occur in colon tumors. The transfection of human gastric MKN-1 cells with miR-145 resulted in a greater growth inhibitory effect than that with miR-143, results which were contrary to those in colon cancers. In MKN-1 cells, an additive effect on growth inhibition was shown by the combined transfection with miR-143 and miR-145; further, higher sensitivity to 5-fluorouracil was also observed following the transfection with miR-143 or miR-145. The possible candidate target messenger RNAs of miR-145 were identified to be insulin receptor substrate-1 and beta-actin. CONCLUSION: Taken together, these findings suggest that miR-143 and miR-145 act as anti-oncomirs common to gastrointestinal tumors.

Chamber-specific differentiation of Nkx2.5-positive cardiac precursor cells from murine embryonic stem cells.

Embryonic stem (ES) cells are a useful system to study cardiac differentiation in vitro. It has been difficult, however, to track the fates of chamber-specific cardiac lineages, since differentiation is induced within the embryoid body. We have established an in vitro culture system to track Nkx2.5(+) cell lineages during mouse ES cell differentiation by using green fluorescent protein (GFP) as a reporter. Nkx2.5/GFP(+) cardiomyocytes purified from embryoid bodies express sarcomeric tropomyosin and myosin heavy chain and heterogeneously express cardiac troponin I (cTnI), myosin light chain 2v (MLC2v) and atrial natriuretic peptide (ANP). After 4-week culture, GFP(+) cells exhibited electrophysiological characteristics specific to sinoatrial (SA) node, atrial, or ventricular type. Furthermore, we found that administration of 10(-7) M retinoic acid (RA) to embryoid bodies increased the percentage of MLC2v(-)ANP(+) cells; this also increased the expression of atrial-specific genes in the Nkx2.5/GFP(+) fraction, in a time- and dose-dependent fashion. These results suggest that Nkx2.5(+) lineage cells possess the potential to differentiate into various cardiomyocyte cell types and that RA can modify the differentiation potential of Nkx2.5(+) cardiomyocytes at an early stage.

Molecular hydrogen attenuates fatty acid uptake and lipid accumulation through downregulating CD36 expression in HepG2 cells.

BACKGROUND: There is accumulating evidence that obesity is closely associated with an impaired free fatty acid metabolism as well as with insulin resistance and inflammation. Excessive fatty acid uptake mediated by fatty acid translocase CD36 plays an important role in hepatic steatosis. Molecular hydrogen has been shown to attenuate oxidative stress and improve lipid, glucose and energy metabolism in patients and animal models of hepatic steatosis and atherosclerosis, but the underlying molecular mechanisms remain largely unknown. METHODS: Human hepatoma HepG2 cells were exposed to palmitate-BSA complex after treatment with or without hydrogen for 24 h. The fatty acid uptake was measured by using spectrofluorometry and the lipid content was detected by Oil Red O staining. JNK phosphorylation and CD36 expression were analyzed by Western blot and real-time PCR analyses. RESULTS: Pretreatment with hydrogen reduced fatty acid uptake and lipid accumulation after palmitate overload in HepG2 cells, which was associated with inhibition of JNK activation. Hydrogen treatment did not alter CD36 mRNA expression but reduced CD36 protein expression. CONCLUSION: Hydrogen inhibits fatty acid uptake and lipid accumulation through the downregulation of CD36 at the protein level in hepatic cultured cells, providing insights into the molecular mechanism underlying the hydrogen effects in vivo on lipid metabolism disorders.

Ethanolic extracts of Brazilian red propolis increase ABCA1 expression and promote cholesterol efflux from THP-1 macrophages.

The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by regulating the cellular efflux of cholesterol. Since ABCA1 plays a pivotal role in cholesterol homeostasis and HDL metabolism, identification of a novel substance that is capable of increasing its expression would be beneficial for the prevention and therapy of atherosclerosis. In the present study, we studied the effects of ethanolic extracts of Brazilian red propolis (EERP) on ABCA1 expression and cholesterol efflux in THP-1 macrophages. EERP enhanced PPARγ and liver X receptor (LXR) transcriptional activity at 5-15µg/ml, which was associated with upregulation of PPARγ and LXRα expression. It was also found that EERP increase the activity of the ABCA1 promoter, which is positively regulated by LXR. Consistent with these findings, treatment with EERP increased both mRNA and protein expression of ABCA1. Finally, EERP upregulated ApoA-I-mediated cholesterol efflux. Our results showed that EERP promote ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression via induction of PPARγ/LXR.

Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells.

MiR-34a was identified as one of the down-regulated micro-RNAs (miRs) in human colorectal cancer 5-fluorouracil (5-FU)-resistant DLD-1 cells compared with those in the parental DLD-1 cells. Exposure to 5-FU at 30 µM activated phosphoinositide 3-kinase (PI3K)/Akt signaling markedly from 12h up to 48 h in the 5-FU-resistant cells compared with that in the parental cells and resulted in an overt difference in growth at those times. Furthermore, the expression of miR-34a in the 5-FU-resistant cells was sustained at a low-level, whereas it was up-regulated in the parental cells after the 5-FU treatment. Sirt1, which is one of the target genes for miR-34a and related to drug-resistance, was strikingly up-regulated in the 5-FU-resistant cells. The ectopic expression of miR-34a in the 5-FU-resistant cells inhibited growth, as in the parental cells, and attenuated the resistance to 5-FU through the down-regulation of Sirt1 and E2F3. Moreover, the silencing of Sirt1 significantly canceled the resistance to 5-FU in the 5-FU-resistant cells. These findings suggest that miR-34a targeting the Sirt1 and E2F3 genes could negatively regulate, at least in part, the resistance to 5-FU in human colorectal cancer DLD-1 cells.

Ethanolic extracts of Brazilian red propolis promote adipocyte differentiation through PPARγ activation.

AIM OF THE STUDY: The aim of present study was to investigate the effects of ethanolic extracts of red propolis (EERP) on adipogenesis and evaluate the molecular basis for their anti-obesity effects. MATERIALS AND METHODS: We tested whether EERP alone could induce differentiation of 3T3-L1 cells, regulate the expression of adipocyte-specific genes and reverse inhibitory effects of TNF-α on their differentiation. Next, we performed a luciferase reporter gene assay to test whether EERP could enhance transcriptional activities of PPARγ and adiponectin promoter activities. RESULTS: EERP strongly induced differentiation of 3T3-L1 preadipocytes into adipocytes, and enhanced the PPARγ transcriptional activity and adiponectin promoter activity. In addition, EERP attenuated the inhibitory effect of TNF-α on adipocyte differentiation and adiponectin production in mature adipocytes. CONCLUSION: The present study indicates that EERP enhance differentiation of 3T3-L1 adipocytes in part by its potency of PPARγ activation and are capable of reversing inhibitory effects of TNF-α on adipocyte differentiation and adiponectin expression. These results suggest the value of EERP as a diet supplement for prevention and treatment of obesity and obesity-associated disorders.

Role of microRNA-143 in Fas-mediated apoptosis in human T-cell leukemia Jurkat cells.

Treatment of Jurkat T cells with Fas-activating antibody (CH-11) facilitated rapid cell death that was shown to be caspase-dependent apoptosis. The expression of miR-143 was up-regulated during the apoptosis with time. The increased expression of miR-143 emerged from 1 to 2h after the treatment, at which time the caspases-8 and -3 were also activated; and this increase was almost canceled by the pretreatment with an inhibitor of caspase-3 or -8. Furthermore, the transfection of Jurkat cells with mature miR-143 induced a significant growth suppression and enhancement of CH-11-induced apoptosis. On the contrary, an extracellular signal-regulated protein kinase 5 (ERK5), which was determined to be a target of miR-143 in colon cancer DLD-1 cells, was time-dependently down-regulated at the translational level after the treatment. During the apoptosis, the expression level of FasL was maintained and the level of nuclear-Foxo3a was increased in the early phase. These data suggest that the up-regulation of miR-143 could be related to the apoptosis in part by targeting ERK5, which leads to promotion of Foxo3a/FasL positive feedback loop.

Wnt11 facilitates embryonic stem cell differentiation to Nkx2.5-positive cardiomyocytes.

Wnt signaling plays a crucial role in the control of morphogenesis in several tissues. Herein, we describe the role of Wnt11 during cardiac differentiation of embryonic stem cells. First, we examined the expression profile of Wnt11 during the course of differentiation in embryoid bodies, and then compared its expression in retinoic acid-treated embryoid bodies with that in untreated. In differentiating embryoid bodies, Wnt11 expression rose along with that of Nkx2.5 expression and continued to increase. When the embryoid bodies were treated with retinoic acid, Wnt11 expression decreased in parallel with the decreased expression of cardiac genes. Further, treatment of embryoid bodies with medium containing Wnt11 increased the expression of cardiac marker genes. Based on these results, we propose that Wnt11 plays an important role for cardiac development by embryoid bodies, and may be a key regulator of cardiac muscle cell proliferation and differentiation during heart development.

Gene expression of FK506-binding proteins 12.6 and 12 during chicken development.

FKBPs are cytosolic receptors for the immunosuppressive drug FK506. FKBP12.6 and FKBP12 associate with cardiac and skeletal muscle isoforms of ryanodine receptors and thereby regulate intracellular Ca(2+) release. The amino acid sequences of FKBP12.6 and FKBP12 are highly conserved among mammals and chicken. The expression profiles of FKBP12.6 and FKBP12 genes during chicken development were compared by Northern blot and in situ hybridization analyses. Transcripts of the FKBP12 gene were abundant throughout the embryo at early stages of development but subsequently underwent gradual down-regulation. Expression of the FKBP12.6 gene was mostly restricted to the heart during early embryogenesis and also underwent subsequent down-regulation, becoming localized to the atrium after hatching. Treatment of early embryos with FK506 had no apparent effect on embryogenesis. Retinoic acid induced marked abnormalities in cardiogenesis, but it did not affect FKBP gene expression. These results suggest that, even though FKBP12.6 and FKBP12 genes are expressed in chick embryos, FK506-sensitive functions of the encoded proteins do not appear to contribute to early embryogenesis or cardiogenesis.

Heart-selective expression of the chicken FK506-binding protein (FKBP) 12.6 gene during embryonic development.

FKBP12.6, a member of the family of FK506-binding proteins, selectively associates with the cardiac isoform of the ryanodine receptor and thereby stabilizes this Ca(2+) release channel. A chicken FKBP12.6 (chFKBP12.6) cDNA was cloned and shown to encode a protein of 108 amino acids. The deduced amino acid sequence of chFKBP12.6 is 91-92% identical to those of mammalian FKBP12.6 proteins. Northern blot analysis revealed that chFKBP12.6 mRNA is largely restricted to the heart during embryonic development and that the abundance of this mRNA in the heart decreases, and it becomes restricted to the atrium during cardiogenesis. In situ hybridization revealed that chFKBP12.6 mRNA is localized to the precardiac mesoderm before formation of the primitive heart tube. Expression of the chFKBP12.6 gene was initially apparent throughout the developing multichambered heart but became restricted to the atria before hatching. Reverse transcription and polymerase chain reaction analysis demonstrated that chFKBP12.6 mRNA is present in the embryo from early gastrulation and is most abundant immediately after the onset of the heartbeat. These observations suggest that the chFKBP12.6 gene is expressed before heart morphogenesis to play a role in excitation-contraction coupling in cardiomyocytes and that the function of the encoded protein becomes increasingly restricted to the atrium during embryonic development.