Junhao Mao

Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.

To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, we identified Axin as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, showed no effect on the canonical Wnt signaling pathway by itself, but acted synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain functioned as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt caused the translocation of Axin to the membrane and enhanced the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, we conclude that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway.

Acquisition of Granule Neuron Precursor Identity Is a Critical Determinant of Progenitor Cell Competence to Form Shh-Induced Medulloblastoma

Whether the brain tumor medulloblastoma originates from stem cells or restricted progenitor cells is unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted central nervous system (CNS) progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNPs) derive from hGFAP(+) and Olig2(+) rhombic lip progenitors. Hh activation in a spectrum of early- and late-stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors.

Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1.

Wnt proteins transduce their signals through dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3β (GSK), leading to the accumulation of cytosolic β‐catenin and activation of TCF/LEF‐1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF‐1, we investigated the roles of Axin and Frat1 in Wnt‐mediated activation of LEF‐1 in mammalian cells. We found that Dvl interacts with Axin and with Frat1, both of which interact with GSK. Similarly, the Frat1 homolog GBP binds Xenopus Dishevelled in an interaction that requires GSK. We also found that Dvl, Axin and GSK can form a ternary complex bridged by Axin, and that Frat1 can be recruited into this complex probably by Dvl. The observation that the Dvl‐binding domain of either Frat1 or Axin was able to inhibit Wnt‐1‐induced LEF‐1 activation suggests that the interactions between Dvl and Axin and between Dvl and Frat may be important for this signaling pathway. Furthermore, Wnt‐1 appeared to promote the disintegration of the Frat1–Dvl–GSK–Axin complex, resulting in the dissociation of GSK from Axin. Thus, formation of the quaternary complex may be an important step in Wnt signaling, by which Dvl recruits Frat1, leading to Frat1‐mediated dissociation of GSK from Axin.

Dishevelled Proteins Lead to Two Signaling Pathways REGULATION OF LEF-1 AND c-Jun N-TERMINAL KINASE IN MAMMALIAN CELLS

Dishevelled (Dsh/Dvl) proteins are known to mediate Wnt signaling by up-regulating β-catenin levels and stimulating T cell factor (TCF)/LEF-1-dependent transcription. We have identified a new Dvl-mediated signaling pathway in that mouse Dvl proteins, when expressed in COS-7 cells, stimulate c-Jun-dependent transcription activity and the kinase activity of the c-Jun N-terminal kinase (JNK). The DEP domain of Dvl1 is essential for JNK activation. By contrast, all three conserved domains of Dvl, including DIX, PDZ, and DEP, are required for up-regulation of β-catenin and for stimulation of LEF-1-mediated transcription in mammalian cells. Thus, Dvl can lead to two different signaling pathways. Furthermore, the small G proteins of Cdc42 or Rac1, which are involved in JNK activation by many stimuli, do not appear to play a major role in Dvl-mediated JNK activation, because the dominant negative mutants of Cdc42 and Rac1 could not inhibit Dvl-induced JNK activation. This suggests that Dvl may activate JNK via novel pathways.

A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway

We report a novel mouse model for the generation of sporadic tumors and show the efficiency of this approach by surveying Hedgehog (Hh)-related tumors. Up-regulation of the Hh pathway is achieved by conditionally regulated expression of an activated allele of Smoothened (R26-SmoM2) using either sporadic leakage or global postnatal induction of a ubiquitously expressed inducible Cre transgene (CAGGS-CreER). Following postnatal tamoxifen induction, CAGGS-CreER; R26-SmoM2 mice developed tumors with short latency and high penetrance. All mice exhibited rhabdomyosarcoma and basal cell carcinoma; 40% also developed medulloblastoma. In addition, mice showed a novel pancreatic lesion resembling low-grade mucinous cystic neoplasms in humans. In contrast, widespread activation of SmoM2 in the postnatal prostate epithelium results in no detectable morphologic outcome in 12-month-old mice. Comparison of gene expression profiles among diverse tumors identified several signature genes, including components of platelet-derived growth factor and insulin-like growth factor pathways, which may provide a common mechanistic link to the Hh-related malignancies. This experimental model provides a robust tool for exploring the process of Hh-dependent tumorigenesis and the treatment of such tumors. More generally, this approach provides a genetic platform for identifying tumorigenic potential in putative oncogenes and tumor suppressors and for more effective modeling of sporadic cancers in mice.

Specific Involvement of G Proteins in Regulation of Serum Response Factor-mediated Gene Transcription by Different Receptors

Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G protein-coupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking Gαq and Gα11. We found that the constitutively active forms of the α subunits of the Gqand G12 class of G proteins, including Gαq, Gα11, Gα14, Gα16, Gα12, and Gα13, can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and α1-adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on Gαq/11, while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of Gαq/11. Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit Gα12 and Gα13-mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin- and LPA-mediated SRF activation in the Gαq/11-deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to Gα12/13. On the contrary, receptors including β2- and α2-ARs, m2R, the dopamine receptors type 1 and 2, angiotensin receptors types 1 and 2, and interleukin-8 receptor could not activate SRF in the presence or absence of Gαq/11, suggesting that these receptors cannot couple to endogenous G proteins of the G12 or Gqclasses.

The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd.

ABSTRACT The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine Wnt.

Hedgehog Signaling Is Dispensable for Adult Murine Hematopoietic Stem Cell Function and Hematopoiesis

We report the unexpected finding that loss of Hh signaling through conditional deletion of Smoothened (Smo) in the adult hematopoietic compartment has no apparent effect on adult hematopoiesis, including peripheral blood count, number or cell-cycle status of stem or progenitor cells, hematopoietic colony-forming potential, long-term repopulating activity in competitive repopulation assays, or stress response to serial 5-fluorouracil treatment. Furthermore, pharmacologic inhibition of Hh signaling with a potent and selective small molecule antagonist has no substantive effect on hematopoiesis in the mouse. In addition, Hh signaling is not required for the development of MLL-AF9-mediated acute myeloid leukemia (AML). Taken together, these data demonstrate that Hh signaling is dispensable for normal hematopoietic development and hematopoietic stem cell function, indicating that targeting of Hh signaling in solid tumors is not likely to result in hematopoietic toxicity. Furthermore, the Hh pathway may not be a compelling target in certain hematopoietic malignancies.

Regulation of Gli1 Transcriptional Activity in the Nucleus by Dyrk1

To investigate the cellular role of dual specificity Yak1-related kinase (Dyrk) 1, a nuclear localized dual specificity protein kinase, we examined its effect on transcriptional regulation using reporter gene assays. We found that Dyrk1 can substantially enhance Gli1-dependent, but not LEF-1-, c-Jun-, or Elk-dependent, gene transcription. In part, Dyrk1 does this through retaining Gli1 in the nucleus. However, we also demonstrate that Dyrk1 can enhance the transcriptional activity of Gli1-AHA, a nuclear export mutant, suggesting that Dyrk1 may be more directly involved in regulating the transcriptional activity of Gli1. In addition, Dyrk1 acted synergistically with Sonic hedgehog (Shh) to induce gene transcription and differentiation in mouse C3H10T1/2 cells. The failure of Shh to stimulate Dyrk1 kinase activity suggests that Dyrk1 may not be directly regulated by the Shh signaling pathway but functionally interacts with it. Thus, Gli1 transcriptional activity may be subjected to further regulation in the cell nucleus by a pathway distinct from Shh signaling, one mediated by Dyrk1.

Modeling Recent Human Evolution in Mice by Expression of a Selected EDAR Variant

An adaptive variant of the human Ectodysplasin receptor, EDARV370A, is one of the strongest candidates of recent positive selection from genome-wide scans. We have modeled EDAR370A in mice and characterized its phenotype and evolutionary origins in humans. Our computational analysis suggests the allele arose in central China approximately 30,000 years ago. Although EDAR370A has been associated with increased scalp hair thickness and changed tooth morphology in humans, its direct biological significance and potential adaptive role remain unclear. We generated a knockin mouse model and find that, as in humans, hair thickness is increased in EDAR370A mice. We identify new biological targets affected by the mutation, including mammary and eccrine glands. Building on these results, we find that EDAR370A is associated with an increased number of active eccrine glands in the Han Chinese. This interdisciplinary approach yields unique insight into the generation of adaptive variation among modern humans.