👤 Pengcheng He

Mutations in the TSC1 and TSC2 genes lead to tuberous sclerosis complex (TSC), which is characterized clinically by mental retardation, epilepsy, and benign tumors affecting multiple tissues. Numerous components of the TSC protein complex remain uncharacterized. Here we report the purification of the TSC1 complex under physiological conditions using a proteomic strategy. We purified the TSC1 protein complex using a tandem affinity purification method and identified a protein complex containing 139 components. Two known binding proteins of TSC1 (TSC2 and DOCK7) were identified along with other new potential partners, which cover reported and novel TSC1 functional categories. Bioinformatics and biochemical methods were used to evaluate the observed protein-protein interactions. A comparative analysis with a published expression proteomics/genomics study of TSC1 revealed more than 20 common candidates that might be functionally relevant. The data set provides new directions in which to expand our knowledge of the functions of TSC1 and the mechanisms of TSC. The results are highly reliable, which is reflected by the identification of a few reported partners of TSC1 and many TSC1/2-regulated proteins. Interestingly, many new functional categories were identified, such as DNA repair, which provide novel hints to the function of TSC1. Moreover, a few neuronal disease-related proteins that might regulate the normal functions of neurons were identified. Thus, the results suggest that many of the new interactions should be biologically significance. It will be interesting to further investigate the regulatory mechanisms of these components. Show less

The identification of estrogen receptors alpha and beta and aromatase in the testis has highlighted the important role of estrogens in regulating spermatogenesis. There is a wealth of information on the deleterious effects of fetal and neonatal exposure of estrogens and xenoestrogens in the testis, including spermiation failure and germ cell apoptosis. However, very little is known about gene transcripts affected by exogenous estradiol exposure in the testis. The objective of the present study was to unveil global gene expression profiles and testicular cell number changes in rats after estradiol treatment. 17beta-estradiol was administered to adult male rats at a dose of 100 micrograms/kg body weight in saline daily for 10 days; male rats receiving only saline were used as controls. Microarray analysis was performed to examine global gene expression profiles with or without estradiol treatment. Real time RT-PCR was conducted to verify the microarray data. In silico promoter and estrogen responsive elements (EREs) analysis was carried out for the differentially expressed genes in response to estradiol. Quantitation of testicular cell number based on ploidy was also performed using flow cytometry in rats with or without estradiol treatment. We found that 221 genes and expressed sequence tags (ESTs) were differentially expressed in rat testes treated with estradiol compared to the control; the microarray data were confirmed by real time RT-PCR. Gene Ontology analysis revealed that a number of the differentially expressed genes are involved in androgen and xenobiotic metabolism, maintenance of cell cytoskeleton, endocytosis, and germ cell apoptosis. A total of 33 up-regulated genes and 67 down-regulated genes showed the presence of EREs. Flow cytometry showed that estradiol induced a significant decrease in 2n cells (somatic and germ cells) and 4n cells (pachytene spermatocytes) and a marked increase in the number of elongated and elongating spermatids. This study provides a novel insight into the molecular basis for spermiation failure and apoptosis caused by 17beta-estradiol and it also offers new mechanisms by which adult exposure to environmental estrogens can affect spermatogenesis and fertility. Show less

Spermatogenesis in man starts with spermatogonial stem cells (SSCs), and leads to the production of sperm in approximately 64 days, common to old and young men. Sperm from elderly men are functional and able to fertilize eggs and produce offspring, even though daily sperm production is more than 50% lower and damage to sperm DNA is significantly higher in older men than in those who are younger. Our hypothesis is that the SSC/spermatogonial progenitors themselves age. To test this hypothesis, we studied the gene expression profile of mouse SSC/progenitor cells at several ages using microarrays. After sequential enzyme dispersion, we purified the SSC/progenitors with immunomagnetic cell sorting using an antibody to GFRA1, a known SSC/progenitor cell marker. RNA was isolated and used for the in vitro synthesis of amplified and labeled cRNAs that were hybridized to the Affymetrix mouse genome microarrays. The experiments were repeated twice with different cell preparations, and statistically significant results are presented. Quantitative RT-PCR analysis was used to confirm the microarray results. Comparison of four age groups (6 days, 21 days, 60 days, and 8 months old) showed a number of genes that were expressed specifically in the older mice. Two of them (i.e. Icam1 and Selp) have also been shown to mark aging hematopoietic stem cells. On the other hand, the expression levels of the genes encoding the SSC markers Gfra1 and Plzf did not seem to be significantly altered by age, indicating that age affects only certain SSC/progenitor properties. Show less

This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation. Show less

Non-steroidal compounds that inhibit 17beta-hydroxysteroid dehydrogenase isoform 3 (17beta-HSD3), an enzyme catalyzing the final step in testosterone biosynthesis in Leydig cells, are under development for male contraceptive or treatment of androgen dependent diseases including prostate cancer. A series of curcumin analogues with more stable chemical structures were compared to curcumin as inhibitors of 17beta-HSD3 in rat intact Leydig cells as well as rat and human testis microsomes. Show less

The microtubule-actin crosslinking factor 1 (MACF1) is a ubiquitous cytoskeletal linker protein with multiple spliced isoforms expressed in different tissues. The MACF1a isoform contains microtubule and actin-binding regions and is expressed at high levels in the nervous system. Macf1-/- mice are early embryonic lethal and hence the role of MACF1 in the nervous system could not be determined. We have specifically knocked out MACF1a in the developing mouse nervous system using Cre/loxP technology. Mutant mice died within 24-36h after birth of apparent respiratory distress. Their brains displayed a disorganized cerebral cortex with a mixed layer structure, heterotopia in the pyramidal layer of the hippocampus, disorganized thalamocortical and corticofugal fibers, and aplastic anterior and hippocampal commissures. Embryonic neurons showed a defect in traversing the cortical plate. Our data suggest a critical role for MACF1 in neuronal migration that is dependent on its ability to interact with both microfilaments and microtubules. Show less

Thioredoxin-interacting protein (Txnip) has important functions in regulating cellular metabolism including glucose utilization; the expression of the Txnip gene is sensitive to the availability of glucose and other fuels. Here, we show that Txnip expression is down-regulated at the transcriptional level by diverse inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). The effect of these OXPHOS inhibitors is mediated by earlier identified carbohydrate-response elements (ChoREs) on the Txnip promoter and the ChoRE-associated transcription factors Max-like protein X (MLX) and MondoA (or carbohydrate-response element-binding protein (ChREBP)) involved in glucose-induced Txnip expression, suggesting that inhibited oxidative phosphorylation compromises glucose-induced effects on Txnip expression. We also show that the OXPHOS inhibitors repress the Txnip transcription most likely by inducing the glycolytic rate, and increased glycolytic flux decreases the levels of glycolytic intermediates important for the function of MLX and MondoA (or ChREBP). Our findings suggest that the Txnip expression is tightly correlated with glycolytic flux, which is regulated by oxidative phosphorylation status. The identified link between the Txnip expression and glycolytic activity implies a mechanism by which the cellular glucose uptake/homeostasis is regulated in response to various metabolic cues, oxidative phosphorylation status, and other physiological signals, and this may facilitate our efforts toward understanding metabolism in normal or cancer cells. Show less

The lipid kinase PIK3C3 (also called Vps34) regulates both the endosomal and autophagic pathways. However, the effect of inactivating PIK3C3 on neuronal endosomal versus autophagic processes in vivo has not been studied. We generated mice in which Pik3c3 was conditionally deleted in differentiated sensory neurons. Within a few days after Pik3c3 deletion, mutant large-diameter myelinated neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated more slowly. These differential degenerative phenotypes are unlikely caused by a disruption in the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct phenotype in all sensory neurons (i.e., formation of very large intracellular inclusion bodies and slow degeneration over a period of several months). More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. Analyses of Pik3c3/Atg7 double mutant neurons revealed that this unconventional initiation pathway still depends on ATG7. Our studies represent in vivo characterization of PIK3C3 functions in mammals and provide insights into the complexity of neuronal endo-lysosomal and autophagic pathways. Show less

To identify loci for age at menarche, we performed a meta-analysis of 32 genome-wide association studies in 87,802 women of European descent, with replication in up to 14,731 women. In addition to the known loci at LIN28B (P = 5.4 × 10⁻⁶⁰) and 9q31.2 (P = 2.2 × 10⁻³³), we identified 30 new menarche loci (all P < 5 × 10⁻⁸) and found suggestive evidence for a further 10 loci (P < 1.9 × 10⁻⁶). The new loci included four previously associated with body mass index (in or near FTO, SEC16B, TRA2B and TMEM18), three in or near other genes implicated in energy homeostasis (BSX, CRTC1 and MCHR2) and three in or near genes implicated in hormonal regulation (INHBA, PCSK2 and RXRG). Ingenuity and gene-set enrichment pathway analyses identified coenzyme A and fatty acid biosynthesis as biological processes related to menarche timing. Show less

To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatography (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejection (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection. Show less

Spermatogenesis is the process that involves the division and differentiation of spermatogonial stem cells into spermatozoa. However, the autocrine molecules and signaling pathways controlling their fate remain unknown. This study was designed to identify novel growth factors and signaling pathways that regulate proliferation, differentiation, and survival of spermatogonial stem/progenitor cells. To this end, we have for the first time explored the expression, function, and signaling pathway of Nodal, a member of the transforming growth factor-beta superfamily, in mouse spermatogonial stem/progenitor cells. We demonstrate that both Nodal and its receptors are present in these cells and in a spermatogonial stem/progenitor cell line (C18-4 cells), whereas Nodal is undetected in Sertoli cells or differentiated germ cells, as assayed by reverse transcription-polymerase chain reaction, Western blots, and immunocytochemistry. Nodal promotes proliferation of spermatogonial stem/progenitor cells and C18-4 cells, whereas Nodal receptor inhibitor SB431542 blocks their propagation as shown by proliferation and bromodeoxyuridine incorporation assays. Nodal knockdown by RNA interference results in a marked increase of cell apoptosis and a reduction of cell division as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling and proliferation assays. Conversely, overexpression of Nodal leads to an increase of cell proliferation. Nodal activates Smad2/3 phosphorylation, Oct-4 transcription, cyclin D1, and cyclin E expression, whereas SB431542 completely abolishes their increase. Together, Nodal was identified as the first autocrine signaling molecule that promotes proliferation of mouse spermatogonial stem/progenitor cells via Smad2/3 and Oct-4 activation. This study thus provides novel and important insights into molecular mechanisms regulating proliferation and survival of spermatogonial stem/progenitor cells. Show less

Small RNA molecules (small RNAs), including small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs), have recently emerged as important regulators of gene expression at the post-transcriptional or translation level. Significant progress has recently been made utilizing small RNAs in elucidating the molecular mechanisms regulating spermatogenesis. Spermatogenesis is a complex process that involves the division and eventual differentiation of spermatogonial stem cells into mature spermatozoa. The process of spermatogenesis is composed of several phases: mitotic proliferation of spermatogonia to produce spermatocytes; two meiotic divisions of spermatocytes to generate haploid round spermatids; and spermiogenesis, the final phase that involves the maturation of early-round spermatids into elongated mature spermatids. A number of miRNAs are expressed abundantly in male germ cells throughout spermatogenesis, while piRNAs are only present in pachytene spermatocytes and round spermatids. In this review, we first address the synthesis, mechanisms of action, and functions of siRNA, miRNA, and piRNA, and then we focus on the recent advancements in defining the small RNAs in the regulation of spermatogenesis. Concerns pertaining to the use of siRNAs in exploring spermatogenesis mechanisms and open questions in miRNAs and piRNAs in this field are highlighted. The potential applications of small RNAs to male contraception and treatment for male infertility and testicular cancer are also discussed. Show less

Spermatogonial stem cells (SSCs) have unique characteristics in that they produce sperm that transmit genetic information from generation to generation and they can be reprogrammed spontaneously to form embryonic stem (ES)-like cells to acquire pluripotency. In rodents, it is generally believed that the A-single (A(s)) is the stem cell population, whereas the A-paired (A(pr)) and A-aligned (A(al)) represent the progenitor spermatogonial population. The A(1) to A(4) cells, intermediate, and type B spermatogonia are considered differentiated spermatogonia. In human, very little information is available about SSCs, except for the earlier work of Clermont and colleagues who demonstrated that there are two different types of A spermatogonia, the A(dark) and A(pale) spermatogonia. The A(dark) spermatogonia were referred to as the reserve stem cells, whereas the A(pale) were considered the renewing stem cells. In this review, we outline several spermatogonial renewal schemes for both rodents and primates, including man. We also compare phenotypic markers for spermatogonia/spermatogonial stem cells in rodents and humans and address SSC potential and therapeutic application. Show less

Spermatogenesis is the process that involves the division and differentiation of spermatogonial stem cells (SSCs) into mature spermatozoa. SSCs are a subpopulation of type A spermatogonia resting on the basement membrane in the mammalian testis. Self-renewal and differentiation of SSCs are the foundation of normal spermatogenesis, and thus a better understanding of molecular mechanisms and signaling pathways in the SSCs is of paramount importance for the regulation of spermatogenesis and may eventually lead to novel targets for male contraception as well as for gene therapy of male infertility and testicular cancer. Uncovering the molecular mechanisms is also of great interest to a better understanding of SSC aging and for developing novel therapeutic strategies for degenerative diseases in view of the recent work demonstrating the pluripotent potential of the SSC. Progress has recently been made in elucidating the signaling molecules and pathways that determine cell fate decisions of SSCs. In this review, we first address the morphological features, phenotypic characteristics, and the potential of SSCs, and then we focus on the recent advances in defining the key signaling molecules and crucial signaling pathways regulating self-renewal and differentiation of SSCs. The association of aberrant expression of signaling molecules and cascades with abnormal spermatogenesis and testicular cancer are also discussed. Finally, we point out potential future directions to pursue in research on signaling pathways of SSCs. Show less

Recent reports have demonstrated that adult cells can be reprogrammed to pluripotency, but mostly with genes delivered using retroviruses. Some of the genes are cancer causing; thus, these adult-derived embryonic stem (ES)-like cells cannot be used for therapy to cure human diseases. Remarkably, it has also been demonstrated recently by several groups that, in mice, spermatogonial stem cells (SSCs) can be reprogrammed to ES-like cells without the necessity of exogenously added genes. SSCs constitute one of the most important stem cell systems in the body, not only because they produce spermatozoa that transmit genetic information from generation to generation, but also because of the recent studies showing their remarkable plasticity. Very little is known about SSCs in humans, except for the earlier work of Clermont and colleagues who demonstrated that there are A(dark) and A(pale) spermatogonia, with the A(dark) referred to as the reserve stem cells and the A(pale) being the renewing stem cells. We now demonstrate that G protein-coupled receptor 125 (GPR125) may be a marker for human SSCs. Putative human SSCs can also be reprogrammed to pluripotency. We were able to achieve this result without the addition of genes, suggesting that human SSCs have considerable potential for cell-based, autologous organ regeneration therapy for various diseases. Show less

To characterize the molecular phenotype of spermatogonial stem cells (SSCs), we examined genes that are differentially expressed in the stem/progenitor spermatogonia compared to nonstem spermatogonia. We isolated type A spermatogonia (stem and nonstem type A) from 6-day-old mice using sedimentation velocity at unit gravity and further selected the stem/progenitor cell subpopulation by magnetic activated cell sorting with an antibody to GDNF-receptor-alpha-1 (GFRA1). It has been previously shown that GFRA1 is expressed in SSCs and is required for their stemness. The purity of the isolated cells was approximately 95% to 99% as indicated by immunocytochemistry using anti-GFRA1. Comparison of GFRA1-positive and GFRA1-negative spermatogonia by microarray analysis revealed 99 known genes and 12 uncharacterized transcripts that are overexpressed in the former cell population with a >2-fold change. Interestingly, the highest level of overexpression was observed for Csf1r, encoding the receptor for macrophage colony-stimulating factor (M-CSF, official symbol CSF1), which has a well-established role in the regulation of myeloid progenitor cells. Analysis of our microarray data with a bioinformatics software program (Ingenuity Systems) revealed the potential role of various signaling pathways in stem/progenitor spermatogonia and suggested a common pathway for GFRA1 and CSF1R that may lead to their proliferation. Further investigation to test this hypothesis has shown that CSF1 promotes cell proliferation in primary cultures of the isolated type A spermatogonia and in the spermatogonial-derived stem cell line C18-4. Semiquantitative RT-PCR and immunohistochemistry confirmed the previously mentioned microarray data. Collectively, this study provides novel molecular signatures for stem/progenitor spermatogonia and demonstrates a role for CSF1/CSF1R signaling in regulating their proliferation. Show less

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension. Show less

Adenoid cystic carcinoma (ACC) is a slow growing but highly invasive cancer with a high recurrence rate. Id (inhibitor of DNA binding) proteins are dominant regulators of basic helix-loop-helix transcription factors that control malignant cell behavior in many different tissues. This study aimed to identify the potential role of inhibiting DNA binding-1 (Id-1) in human salivary adenoid cystic carcinoma (SACC) progression. First, we compared the Id-1 protein expression in a human salivary adenoid cystic carcinoma cell line (ACCM) against three other cell lines and found that Id-1 protein expression in ACCM to be significantly higher. Then we measured Id-1 mRNA and protein expression in ACCM before and after RNA interference (RNAi), which showed successful inhibition of Id-1. Further studies then demonstrated that the proliferation and invasiveness of ACCM cells were dramatically down-regulated, and increased numbers of apoptotic cells were detected after Id-1 silencing. Consequently, our data suggest that Id-1 is a potential target in the treatment of human salivary adenoid cystic carcinoma. Show less

The liver X receptors (LXRs) have been known as sterol sensors that impact cholesterol and lipid homeostasis, as well as inflammation. Although the hepatic functions of LXRs are well documented, whether and how LXRs play a pathophysiological role in the lung remain largely unknown. Here we show that LXRalpha and LXRbeta are expressed in both type I and type II mouse lung epithelial cells, as well as in human lung cancer cells. To study the role of LXRalpha in vivo including the pulmonary function of this LXR isoform, we created LXRalpha knock-in (LXR-KI) mice in which a constitutively activated LXRalpha (VP-LXRalpha) was inserted into the mouse LXRalpha locus. We show that activation of LXR in LXR-KI mice or LXR agonist-treated wild type mice induced pulmonary expression of genes encoding multiple antioxidant enzymes. Consistent with the induction of antioxidant enzymes, LXR-KI mice and LXR ligand-treated wild type mice showed a substantial resistance to lipopolysaccharide-induced lung injury and decreased production of reactive oxygen species. In summary, we have uncovered a novel role of LXR in regulating antioxidant enzymes in the lung and the implication of this regulation in pulmonary tissue protection. Show less

Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of beta-catenin. Activation of PKA is essential for PTH-induced beta-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of beta-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity. Show less

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors. Show less

Canonical Wnt/beta-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/beta-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/beta-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate beta-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/beta-catenin signaling. Show less

Human malignant melanoma cell line UACC903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line UACC903(+6) is sensitive. Here, we describe identification of differential molecular pathways underlying this difference. Using our recently developed mitochondria-focused cDNA microarrays, we identified 154 differentially expressed genes including proapoptotic (BAK1 [6p21.3], BCAP31, BNIP1, CASP3, CASP6, FAS, FDX1, FDXR, TNFSF10 and VDAC1) and antiapoptotic (BCL2L1, CLN3 and MCL1) genes. Expression of these pro- and anti-apoptotic genes was higher in UACC903(+6) than in UACC903 before UV treatment and was altered after UV treatment. qRT-PCR and Western blots validated microarray results. Our bioinformatic analysis mapped these genes to differential molecular pathways that predict resistance and sensitivity of UACC903 and UACC903(+6) to apoptosis respectively. The pathways were functionally confirmed by the FAS ligand-induced cell death and by siRNA knockdown of BAK1 protein. These results demonstrated the differential molecular pathways underlying survival and apoptosis of UACC903 and UACC903(+6) cell lines. Show less

Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in regulating the proliferation of spermatogonial stem cells (SSC). The signaling pathways mediating the function of GDNF in SSC remain unclear. This study was designed to determine whether GDNF signals via the Ras/ERK1/2 pathway in the C18-4 cells, a mouse SSC line. The identity of this cell line was confirmed by the expression of various markers for germ cells, proliferating spermatogonia, and SSC, including GCNA1, Vasa, Dazl, PCNA, Oct-4, GFRalpha1, Ret, and Plzf. Western blot analysis revealed that GDNF activated Ret tyrosine phosphorylation. All 3 isoforms of Shc were phosphorylated upon GDNF stimulation, and GDNF induced the binding of the phosphorylated Ret to Shc and Grb2 as indicated by immunoprecipitation and Western blotting. The active Ras was induced by GDNF, which further activated ERK1/2 phosphorylation. GDNF stimulated the phosphorylation of CREB-1, ATF-1, and CREM-1, and c-fos transcription. Notably, the increase in ERK1/2 phosphorylation, c-fos transcription, bromodeoxyuridine incorporation, and metaphase counts induced by GDNF, was completely blocked by pretreatment with PD98059, a specific inhibitor for MEK1, the upstream regulator of ERK1/2. GDNF stimulation eventually upregulated cyclin A and CDK2 expression. Together, these data suggest that GDNF induces CREB/ATF-1 family member phosphorylation and c-fos transcription via the Ras/ERK1/2 pathway to promote the proliferation of SSC. Unveiling GDNF signaling cascades in SSC has important implications in providing attractive targets for male contraception as well as for the regulation of stem cell renewal vs. differentiation. Show less

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis. Show less

Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions. Show less

The PIK3C3 gene has been implicated as a candidate gene for schizophrenia by functional evidence and genetic association studies. A series of previous studies have found susceptibility SNPs in promoter region. To further verify its susceptibility to schizophrenia in the Chinese population and the function of the polymorphisms, we performed a case control study in 556 unrelated schizophrenia patients and 563 normal controls as well as an in vitro functional analysis. In our association analysis of-432C-/T, we discovered obvious differences in allele frequency between patients and controls (P=0.017). A T/C haplotype constructed by -432C-/T and -86insC, which are tightly linked with each other (r(2)=1) can significantly weaken promoter's transcriptional activity by 20% (p=0.002 by t-test). Though we cannot exclude the possibility that susceptibility of -432C-/T is caused by its linkage disequilibrium with other causal variants, our results do support PIK3C3 play a significant role in the etiology of schizophrenia. Show less

This study aims to identify the effects of antipsychotics on plasma proteins, and on the proteins associated with schizophrenia. We applied proteomics technology to screen protein aberrations in Sprague-Dawley rats treated with antipsychotics and schizophrenic patients undergoing medication. ApoA-I was found significantly increased in the chlorpromazine-treated rats and decreased in the patients with treatment-resistant schizophrenia, which suggest that decreased levels of apoA-I might be associated with the pathology of schizophrenia and that chlorpromazine increases apoA-I levels as part of its therapeutic action. Show less

Constitutive activation of the Wnt pathway as a result of APC, AXIN1 or CTNNB1 mutations has been found in most colorectal cancers. For a long time, this aberrant Wnt activation has been thought to be independent of upstream signals. However, recent studies indicate that upstream signals retain their ability to regulate the Wnt pathway even in the presence of downstream mutations. Wnt-2 is well known for its overexpression in colorectal cancer. Galectin-3 (Gal-3), a multifunctional carbohydrate binding protein implicated in a variety of biological functions, has recently been reported to interact with beta-catenin. In this study, we investigated roles of Wnt-2 and Gal-3 in the regulation of canonical Wnt/beta-catenin signaling. We found that siRNA silencing of either Wnt-2 or Gal-3 expression inhibited TCF-reporter activity, decreased cytosolic beta-catenin level and induced apoptosis in human colorectal cancer cells containing downstream mutations. More interestingly, we showed that inhibition of both Wnt-2 and Gal-3 had synergistic effects on suppressing canonical Wnt signaling and inducing apoptosis, suggesting that aberrant canonical Wnt/beta-catenin signaling in colorectal cancer can be regulated at multiple levels. The combined inhibition of Wnt-2 and Gal-3 may be of superior therapeutic advantage to inhibition by either one of them, giving rise to a potential development of novel drugs for the targeted treatment of colorectal cancer. Show less