Zhi-Yu Wang

NOTCH inhibits osteoblast formation in inflammatory arthritis via noncanonical NF-κB

NOTCH-dependent signaling pathways are critical for normal bone remodeling; however, it is unclear if dysfunctional NOTCH activation contributes to inflammation-mediated bone loss, as observed in rheumatoid arthritis (RA) patients. We performed RNA sequencing and pathway analyses in mesenchymal stem cells (MSCs) isolated from transgenic TNF-expressing mice, a model of RA, to identify pathways responsible for decreased osteoblast differentiation. 53 pathways were dysregulated in MSCs from RA mice, among which expression of genes encoding NOTCH pathway members and members of the noncanonical NF-κB pathway were markedly elevated. Administration of NOTCH inhibitors to RA mice prevented bone loss and osteoblast inhibition, and CFU-fibroblasts from RA mice treated with NOTCH inhibitors formed more new bone in recipient mice with tibial defects. Overexpression of the noncanonical NF-κB subunit p52 and RELB in a murine pluripotent stem cell line increased NOTCH intracellular domain-dependent (NICD-dependent) activation of an RBPjκ reporter and levels of the transcription factor HES1. TNF promoted p52/RELB binding to NICD, which enhanced binding at the RBPjκ site within the Hes1 promoter. Furthermore, MSC-enriched cells from RA patients exhibited elevated levels of HES1, p52, and RELB. Together, these data indicate that persistent NOTCH activation in MSCs contributes to decreased osteoblast differentiation associated with RA and suggest that NOTCH inhibitors could prevent inflammation-mediated bone loss.

Oligodendrocyte myelin glycoprotein (OMgp) in rat hippocampus is depleted by chronic ethanol consumption.

The hippocampal formation has been shown to be particularly vulnerable to the neurotoxic effects of chronic ethanol consumption. It was hypothesized that this damage was due to the disruption of the expression of neurotrophic factors and certain other proteins within the hippocampus. By using real-time reverse transcription-polymerase chain reaction (RT-PCR) techniques, this study aimed to determine whether chronic ethanol consumption could alter the mRNA expression level of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and oligodendrocyte myelin glycoprotein (OMgp) in the hippocampus. Wistar male rats received an unrestricted access to a liquid diet containing 5% (v/v) ethanol as the sole source of fluid from 10 to 29 weeks of age. Control rats had unlimited access to a liquid diet containing an isocaloric amount of sucrose. We found that chronic ethanol consumption did not cause significant changes in the levels of mRNA for BDNF and GDNF. However, OMgp mRNA showed a significant deficit in ethanol-treated animals. It is suggested that this deficit may be related to the demyelination that is commonly observed in human alcoholics and that this may contribute to the functional and cognitive deficits.

Short-term exposure to ethanol causes a differential response between nerve growth factor and brain-derived neurotrophic factor ligand/receptor systems in the mouse cerebellum.

Alcohol ingestion affects both neuropsychological and motor functions. We hypothesized that one of the key factors involved in such functions are neurotrophins and their receptors. We have therefore examined the effects of short-term ethanol exposure on the mRNA expression and protein levels of neurotrophin ligands and receptors in the cerebellum using real-time RT-PCR and Western blotting techniques. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. The pair-fed control mice were fed an identical liquid diet except that sucrose was substituted isocalorically for ethanol. The cerebellum of mice exhibiting intoxication signs of stage 1 or 2 were used in the present study. We found that exposure to ethanol resulted in elevated levels of nerve growth factor (NGF) and TrkA mRNA expression but a decreased level of brain-derived neurotrophic factor (BDNF) mRNA expression. The expression of TrkB and p73 mRNA was unchanged. Changes in the level of these proteins were found to mirror these mRNA expression levels. We conclude that exposure to ethanol for a short period can cause a differential responsive in the various neurotrophin ligand/receptor systems. The functional consequences of these changes are unknown at present.

Neonatal repetitive maternal separation causes long-lasting alterations in various neurotrophic factor expression in the cerebral cortex of rats.

AIMS This study was carried out to examine the effects of early postnatal maternal separation stress on the development of the cerebral cortex with respect to time-dependent fluctuations of neurotrophic factor ligand and receptor expression. MAIN METHODS Wistar rats were separated from their mothers for 3h per day during postnatal days (PND) 10 to 15. The cerebral cortex was analyzed by real-time RT-PCR for the evaluation of the expression of mRNA for brain-derived neurotrophic factor (BDNF), TrkB, insulin-like growth factor-1 (IGF-1), and type 1 IGF receptor (IGF-1R) on PND16, 20, 30, and 60. KEY FINDINGS The expression of these neurotrophic factor ligands and receptors in the cerebral cortex was enhanced on PND16 and PND20, and then it returned to baseline levels on PND30. By PND60, however, the expression levels were attenuated. SIGNIFICANCE The important implication of this study is the persistent abnormal fluctuation of neurotrophic factor expression for a prolonged period, triggered even after the brain growth spurt. Given that neurotrophic factors play important roles in brain development, it can be speculated that the altered expression of these factors induced by maternal separation may interrupt normal brain development and ultimately lead to functional disruption. However, the possibility of such changes leading to various functional disruptions and the underlying mechanisms involved require further study.

Ethanol neurotoxicity and dentate gyrus development

ABSTRACT  Maternal alcohol ingestion during pregnancy adversely affects the developing fetus, often leading to fetal alcohol syndrome (FAS). One of the most severe consequences of FAS is brain damage that is manifested as cognitive, learning, and behavioral deficits. The hippocampus plays a crucial role in such abilities; it is also known as one of the brain regions most vulnerable to ethanol‐induced neurotoxicity. Our recent studies using morphometric techniques have further shown that ethanol neurotoxicity appears to affect the development of the dentate gyrus in a region‐specific manner; it was found that early postnatal ethanol exposure causes a transitory deficit in the hilus volume of the dentate gyrus. It is strongly speculated that such structural modifications, even transitory ones, appear to result in developmental abnormalities in the brain circuitry and lead to the learning disabilities observed in FAS children. Based on reports on possible factors deciding ethanol neurotoxicity to the brain, we review developmental neurotoxicity to the dentate gyrus of the hippocampal formation.

Direct effects of diethylstilbestrol on the gene expression of the cholesterol side-chain cleavage enzyme (P450scc) in testicular Leydig cells.

AIMS To investigate the precise mechanisms underlying the action of estrogenic endocrine disruptors, we evaluated the direct effects of synthetic estrogen diethylstilbestrol (DES) on steroidogenesis in Leydig cells, with particular emphasis on the expression of the cholesterol side-chain cleavage enzyme P450scc. Furthermore, the mechanism underlying the action of DES was compared with that of endogenous estrogen 17beta-estradiol (E2), which has a potency equivalent to that of DES. MAIN METHODS TTE1 Leydig cells were treated with 5 x 10(-)(8) microM to 5 microM DES or E2 for 24h, and P450scc gene expression and the histone modifications underlying their transcriptional activation were examined using reverse transcription-polymerase chain reaction (RT-PCR) and chromatin immunoprecipitation (ChIP), respectively. KEY FINDINGS P450scc mRNA expression in the DES-treated and E2-treated cells reduced in inverse proportion to the dose of DES and E2, respectively; however, cAMP stimulation induced a recovery in the expression to a level approximately equal to those in the controls. In the DES-treated cells, ChIP assay revealed histone deacetylation in the P450scc promoter region. Interestingly, E2 did not cause histone deacetylation. SIGNIFICANCE In the early stages of steroidogenesis, DES and E2 directly induced a reduction in P450scc mRNA expression in inverse proportion to their doses, and treatment with cAMP restored the decreased P450scc mRNA expression. Furthermore, DES can induce alterations in the histone modification of the P450scc gene, and natural estrogen and synthetic estrogenic compounds such as DES may induce reproductive disorders through different molecular mechanisms.

Ubiquitin E3 Ligase Itch Negatively Regulates Osteoclast Formation by Promoting Deubiquitination of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6

Background: TRAF6 activity is crucial for osteoclastogenesis, which is decreased by deubiquitination. The role of Itch in this process is studied. Results: Itch−/− osteoclast precursors formed more osteoclasts, had prolonged RANKL-induced NF-κB activation, and had delayed TRAF6 deubiquitination. Itch bound to the deubiquitinating enzyme cylindromatosis. Conclusion: Itch inhibits osteoclastogenesis by binding to cylindromatosis and prompting TRAF6 deubiquitination. Significance: Itch is a new inhibitor of osteoclastogenesis. Itch is a ubiquitin E3 ligase that regulates protein stability. Itch−/− mice develop an autoimmune disease phenotype characterized by itchy skin and multiorgan inflammation. The role of Itch in the regulation of osteoclast function has not been examined. We report that Itch−/− bone marrow and spleen cells formed more osteoclasts than cells from WT littermates in response to receptor activator of NF-κB ligand (RANKL) and was associated with increased expression of the osteoclastogenic transcription factors c-fos and Nfatc1. Overexpression of Itch in Itch−/− cells rescued increased osteoclastogenesis. RANKL increased Itch expression, which can be blocked by a NF-κB inhibitor. The murine Itch promoter contains NF-κB binding sites. Overexpression of NF-κB p65 increased Itch expression, and RANKL promoted the binding of p65 onto the NF-κB binding sites in the Itch promoter. Itch−/− osteoclast precursors had prolonged RANKL-induced NF-κB activation and delayed TNF receptor-associated factor 6 (TRAF6) deubiquitination. In WT osteoclast precursors, Itch bound to TRAF6 and the deubiquitinating enzyme cylindromatosis. Adult Itch−/− mice had normal bone volume, but they had significantly increased LPS-induced osteoclastogenesis and bone resorption. Thus, Itch is a new RANKL target gene that is induced during osteoclastogenesis. Itch interacts with the deubiquitinating enzyme and is required for deubiquitination of TRAF6, thus limiting RANKL-induced osteoclast formation.

Regional differences in vulnerability of the cerebellar foliations of rats exposed to neonatal X-irradiation

The purpose of the present study was to elucidate regional differences in the vulnerability of cerebellar foliations of rats exposed to X-irradiation. Effects of X-irradiation on foliations were examined with respect to histological changes in Purkinje cells and Bergmann glial fibers by calbindin-D28k (CB) and glial fibrillary acidic protein (GFAP) immunohistochemistry, respectively. Wistar rats were exposed to X-irradiation (1.5 Gy) on postnatal day (PND) 1. At 3 weeks of age, the cerebellum was examined. The cerebella of rats exposed to X-irradiation showed smaller and abnormal foliations compared with controls. Fewer cerebellar foliations due to fusion with neighboring folia were observed in folia I-III and VIa-VII. Moreover, the extent of such abnormalities was more severe in the latter folia. CB-immunoreactive (IR) Purkinje cells exhibited thin, short, disoriented dendrites that had invaded the granular layer or white matter. On the other hand, GFAP-IR Bergmann glial fibers had not extended their processes into the molecular layer perpendicular to the pial surface, and they appeared thin and disoriented. Accordingly, the above cerebellar abnormalities were more severe in folia I-III, VIa-VII and X than in other regions. In contrast to the histological alterations in these folia, there were no apparent qualitative differences in folia IV-V between X-irradiated and controls. These findings indicate regional difference in the vulnerability of cerebellar folia to X-irradiation. Such differences might be attributed to the cerebellar neurogenetic gradient.

Early postnatal repeated maternal deprivation causes a transient increase in OMpg and BDNF in rat cerebellum suggesting precocious myelination

Repetitive maternal deprivation (MD) of neonatal rats during early life is known as one of the strongest stressors to pre-weaned animals. There is increasing evidence that the cerebellum is involved in cognition and emotion. In the present study, we examined how neurotrophic factors and myelin-associated molecules and their receptors (NGF, BDNF, OMgp, TrkA, TrkB, p75 NTR, and NgR) in the cerebellum are affected by early postnatal maternal separation. Rat pups were separated from their mothers for 3h/day during postnatal days (PND) 10-15. At PND 16 and 30, the levels of mRNA and protein in the cerebellum were determined using real-time PCR and Western blot analysis. Cerebellar mRNA and protein levels of BDNF, TrkB, and OMgp were significantly increased in MD rats at PND 16. However, by PND 30 these variables normalized to control levels. In contrast, the levels of mRNA and protein for NGF, TrkA, p75 NTR, and NgR were unchanged at both ages examined. Transient enhancement of neurotrophic system and myelin-associated molecule expression may cause interference of normal development of the cerebellum such as precocious myelination, which may lead to functional and cognitive deficits later in life.

Epigenetic abnormality of SRY gene in the adult XY female with pericentric inversion of the Y chromosome

In normal ontogenetic development, the expression of the sex‐determining region of the Y chromosome (SRY) gene, involved in the first step of male sex differentiation, is spatiotemporally regulated in an elaborate fashion. SRY is expressed in germ cells and Sertoli cells in adult testes. However, only few reports have focused on the expressions of SRY and the other sex‐determining genes in both the classical organ developing through these genes (gonad) and the peripheral tissue (skin) of adult XY females. In this study, we examined the gonadal tissue and fibroblasts of a 17‐year‐old woman suspected of having disorders of sexual differentiation by cytogenetic, histological, and molecular analyses. The patient was found to have the 46,X,inv(Y)(p11.2q11.2) karyotype and streak gonads with abnormally prolonged SRY expression. The sex‐determining gene expressions in the patient‐derived fibroblasts were significantly changed relative to those from a normal male. Further, the acetylated histone H3 levels in the SRY region were significantly high relative to those of the normal male. As SRY is epistatic in the sex‐determination pathway, the prolonged SRY expression possibly induced a destabilizing effect on the expressions of the downstream sex‐determining genes. Collectively, alterations in the sex‐determining gene expressions persisted in association with disorders of sexual differentiation not only in the streak gonads but also in the skin of the patient. The findings suggest that correct regulation of SRY expression is crucial for normal male sex differentiation, even if SRY is translated normally.