👤 Dezhi He

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation. Show less

To explore the disease associated gene mutation of multiple exostoses by family analysis. Polymerase chain reaction and DNA sequencing were used to detect the mutation hot spot regions of EXT1 and EXT2 gene, while restriction fragment length polymorphism was performed to screen the mutation. We found a novel heterozygous mutation c.811T ->C in EXT1 gene of patients, which resulted in the substitution of histidine for tyrosine at codon 271 in this hereditary multiple exostoses family. The mutation was not found in the unaffected family members, nor in the 100 unrelated normal individual, which was unreported before. The novel mutation Y271H is the disease-causing mutation in the hereditary multiple exostoses family. Show less

Schizophrenia is a relatively common psychiatric syndrome that affects virtually all brain functions. We investigated the plasma proteome of 22 schizophrenia male patients and 20 healthy male controls using two-dimensional gel electrophoresis and mass spectrometry. In total, we have identified 66 protein spots in human plasma and found that seven of them showed altered changes in schizophrenia patients, as compared to healthy controls, which mainly were acute phase proteins (APPs). Among these APPs, haptoglobin alpha2 chain (p < 0.001), haptoglobin beta chain (p < 0.001), alpha1-antitrypsin (p = 0.001), and complement factor B precursor (p = 0.022) showed overexpression in schizophrenia patients, whereas apolipoprotein A-I (p = 0.034) and transthyretin (p = 0.035) were found to be significantly decreased in patients. In addition, the expression of apolipoprotein A-IV (p = 0.018) was significantly up-regulated in schizophrenia patients, as compared to controls. We also found these APP genes, which were differentially expressed in this study, overlap in the schizophrenia susceptibility loci. Our findings further support the hypothesis that the inflammatory response system is linked to the pathophysiology of schizophrenia. Show less

Male infertility is now a major reproductive health problem because of an increasing number of environmental pollutants and chemicals, which eventually result in gene mutations. Genetic alterations caused by environmental factors account for a significant percentage of male infertility. Microarray technology is a powerful tool capable of measuring simultaneously the expression of thousands of genes expressed in a single sample. Eventually, advances in genetic technology will allow for the diagnosis of patients with male infertility due to congenital reasons or environmental factors. Since its introduction in 1994, microarray technology has made significant advances in the identification and characterization of novel or known genes possibly correlated with male infertility in mice, as well as in humans. This provides a rational basis for the application of microarray to establishing molecular signatures for the diagnosis and gene therapy targets of male infertility. In this review, the differential gene expression patterns characterized by microarray in germ and somatic cells at different steps of development or in response to stimuli, as well as a number of novel or known genes identified to be associated with male infertility in mice and humans, are addressed. Moreover, issues pertaining to measurement reproducibility are highlighted for the application of microarray data to male infertility. Show less

The small GTPases Rab5 and Rab21 are closely related, and play essential roles in endocytic trafficking. Rab5 is regulated by VPS9-domain-containing guanine nucleotide exchange factors. Here, we describe a new VPS9-domain protein with ankyrin repeats, the VPS9-ankyrin-repeat protein (Varp). Varp interacts preferentially with GDP-bound Rab21 and has a much stronger guanine nucleotide exchange activity towards Rab21 than Rab5. Furthermore, RNAi-mediated depletion of endogenous Varp significantly disrupts the activity of Rab21 in HeLa cells. Ectopically expressed Varp mainly localizes to early endosomes and causes enlargement of early endosomes and giant late endosomes. Both the VPS9 domain and ankyrin-repeats are required for the endosomal localization and the activity of Varp in vivo. These results suggest that Varp is a potential Rab21 guanine nucleotide exchange factor and might regulate endosome dynamics in vivo. Show less

Signalling by the Wnt family of secreted lipoproteins has essential functions in development and disease. The canonical Wnt/beta-catenin pathway requires a single-span transmembrane receptor, low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6), whose phosphorylation at multiple PPPSP motifs is induced upon stimulation by Wnt and is critical for signal transduction. The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a 'dual-kinase' mechanism for LRP6 phosphorylation and activation. Glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling through the promotion of beta-catenin phosphorylation and degradation, mediates the phosphorylation and activation of LRP6. We show that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1, and this dual phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We show further that a membrane-associated form of GSK3, in contrast with cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt co-receptor activation, reveal an unexpected and intricate logic of Wnt/beta-catenin signalling, and illustrate GSK3 as a genuine switch that dictates both on and off states of a pivotal regulatory pathway. Show less

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis. Show less

The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination. Show less

A major obstacle for successful axon regeneration in the adult central nervous system (CNS) arises from inhibitory molecules in CNS myelin, which signal through a common receptor complex on neurons consisting of the ligand-binding Nogo-66 receptor (NgR) and two transmembrane coreceptors, p75 and LINGO-1. However, p75 expression is only detectable in subpopulations of mature neurons, raising the question of how these inhibitory signals are transduced in neurons lacking p75. In this study, we demonstrate that TROY (also known as TAJ), a TNF receptor family member selectively expressed in the adult nervous system, can form a functional receptor complex with NgR and LINGO-1 to mediate cellular responses to myelin inhibitors. Also, both overexpressing a dominant-negative TROY or presence of a soluble TROY protein can efficiently block neuronal response to myelin inhibitors. Our results implicate TROY in mediating myelin inhibition, offering new insights into the molecular mechanisms of regeneration failure in the adult nervous system. Show less

Clinical researches have shown that there is a genetic contribution to the pathogenesis of schizophrenia. Recent studies have suggested that three genes neuropeptide Y (NPY), phosphoinositide-3-kinase class 3 (PIK3C3) and 14-3-3 eta chain gene (YWHAH) are probably associated with schizophrenia. To replicate these findings, we carried out a family-based study on a sample of 235 trios. Our results suggest that the polymorphisms at the NPY and YWHAH genes are unlikely to be linked with genetic susceptibility to schizophrenia. However, we found significant evidence of preferential transmission of the -432C allele of the PIK3C3 gene in the entire trios (Z=2.91, d.f.=1, P=0.0036) and the male probands trios (Z=2.66, d.f.=1, P=0.0079). Show less

LDL receptor related proteins 5 and 6 (LRP5/6) and their Drosophila homolog Arrow are single-span transmembrane proteins essential for Wnt/beta-catenin signaling, likely via acting as Wnt coreceptors. How Wnt activates LRP5/6/Arrow to initiate signal transduction is not well defined. Here we show that a PPPSP motif, which is reiterated five times in the LRP5/6/Arrow intracellular domain, is necessary and sufficient to trigger Wnt/beta-catenin signaling. A single PPPSP motif, upon transfer to the LDL receptor, fully activates the Wnt pathway, inducing complete axis duplication in Xenopus and TCF/beta-catenin-responsive transcription in human cells. We further show that Wnt signal-ing stimulates, and requires, phosphorylation of the PPPSP motif, which creates an inducible docking site for Axin, a scaffolding protein controlling beta-catenin stability. Our study identifies a critical signaling module and a key phosphorylation-dependent activation step of the Wnt receptor complex and reveals a unifying logic for transmembrane signaling by Wnts, growth factors, and cytokines. Show less

Carbohydrate response element-binding protein (ChREBP) is a rat homolog of human Williams-Beuren syndrome region 14 and a member of the basic helix-loop-helix leucine zipper transcription factor family. Its activation was found to be inducible by carbohydrate in the liver nuclear extracts from rats fed a high-sucrose diet. ChREBP is able to bind to the carbohydrate response element on the promoter of L-type pyruvate kinase and initiate the gene transcription. The detailed expression profile and transcriptional regulation of the ChREBP gene in adipocytes have not been characterized. In the present study, we provide evidence showing that 1) the ChREBP gene is expressed in differentiated 3T3-L1 adipocytes and rat adipose tissue; 2) insulin, glucose, and the antidiabetic agent troglitazone can significantly upregulate the gene expression of ChREBP in 3T3-L1 adipocytes, whereas free fatty acids suppress its expression in this cell type; 3) fasting followed by refeeding with a high-carbohydrate diet resulted in a 10-fold increase of ChREBP mRNA level in rat adipose tissue; and 4) ChREBP expression in adipose tissue is not significantly affected by the diabetic state. Taken together, the results we present are consistent with the idea that ChREBP is an important modulator of adipocyte biology and that its expression in adipose tissue is subject to combined regulation by glucose and insulin in vivo. The induction of ChREBP may serve as a novel pharmacological pathway for troglitazone-mediated hypoglycemic effects in vivo. Show less

We applied proteomics technologies to analyze the cerebrospinal fluid of patients with schizophrenia. Such an analysis can result in the identification of proteins, which may play a role in the disease progress and thus lead to the discovery of clues of the etiology of schizophrenia. Cerebrospinal fluid from patients and controls was analyzed by two-dimensional gels and the proteins were identified by matrix-assisted laser desorption ionization mass spectrometry (MS) in the MS and MS/MS mode. 54 different gene products were identified, which were mainly plasma proteins. The level of apolipoprotein A-IV was significantly decreased in the schizophrenic patients compared to that in the controls. Little is known about the function of this apolipoprotein in the central nervous system. The levels of certain other proteins, like haptoglobin, fibrinogen, complement component 3, and Gc-globulin, were altered in the disease group as well, however, the changes did not reach a statistical significance. Show less

Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes. Show less

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells. Show less

Regulation of beta-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of beta-catenin by the Axin-glycogen synthase kinase (GSK)-3beta complex targets beta-catenin for ubiquitination-degradation, and mutations at critical phosphoserine residues stabilize beta-catenin and cause human cancers. How beta-catenin phosphorylation results in its degradation is undefined. Here we show that phosphorylated beta-catenin is specifically recognized by beta-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. beta-catenin harboring mutations at the critical phosphoserine residues escapes recognition by beta-Trcp, thus providing a molecular explanation for why these mutations cause beta-catenin accumulation that leads to cancer. Inhibition of endogenous beta-Trcp function by a dominant negative mutant stabilizes beta-catenin, activates Wnt/beta-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, beta-Trcp plays a central role in recruiting phosphorylated beta-catenin for degradation and in dorsoventral patterning of the Xenopus embryo. Show less