Co-lead, Cancer Biology Program, Stanford Cancer Center (2012 - Present)
Vice Chair, Department of Medicine (2015 - Present)
Honors & Awards
Maureen Lyles D'Ambrogio Professor of Medicine, Stanford University School of Medicine (2015)
Fellow, AAAS (2015)
Member, American Academy of Physicians (2015)
Consulting Editor, JCI (2012)
American Heart Association Innovative Science Award, AHA (2012)
Research Chair, NIH Intestinal Stem Cell Consortium, NIH (2009)
Transformative R01 Award, NIH (2009)
Member, American Society for Clinical Investigation, American Society for Clinical Investigation (2007)
Samantha Janower Research Chair, Brain Tumor Society (2005)
Merck Faculty Development Award, Merck (2003)
Kimmel Foundation Scholar in Translational Science, Kimmel Foundation (2002)
Burroughs Wellcome Foundation New Investigator in Pharmacological Sciences, Burroughs Wellcome Foundation (2001)
HHMI Physician-Scientist Fellowship, HHMI (1998)
Summa cum laude, Harvard College (1987)
Boards, Advisory Committees, Professional Organizations
Scientific Advisory Board, AP Giannini Foundation (2008 - Present)
Advisory Board, American Heart Association Silicon Valley Chapter (2013 - Present)
A.B., Harvard College, Biochemical Sciences (1987)
M.D./Ph.D., Stanford University, Cancer Biology (1994)
Internship/ Residency, Brigham and Women's Hospital, Internal Medicine (1997)
Fellowship, Dana-Farber/Partners, Adult Oncology (2000)
Current Research and Scholarly Interests
Studying cancer biology and oncogene discovery in organoid cultures.
We have successfully established primary 3D organoid cultures of diverse tissues and used them to achieve the first in vitro conversion of primary intestine, stomach and pancreas tissue to adenocarcinoma (Ootani et al, Nat Med 2009; Li et al, Nat Med 2014) amongst others. These organoid systems comprise an robust in vitro system which we are exploiting for the functional validation of putative oncogenic loci which are identified in whole-genome cancer surveys such as TCGA. We collaborate extensively with systems biologists to interrogate large-scale cancer genomics datasets in organoids using barcoded screening methods, which are identifying new oncogenes against which we then attempt to develop new therapies. We are also applying organoid models to cancer drug discovery and drug resistance.
Intestinal stem/progenitor biology.
The complete regeneration of the epithelial lining of the intestine every 5-7 days renders the intestine a model system for studying stem cell behaviors. We are investigating the regulation of the intestinal stem cell (ISC) compartment by extracellular signals such as Wnts, using adenoviral and conditional knockout approaches. We have defined R-spondins as dominant regulators of the ISC niche with Wnts playing a more permissive role using lineage tracing, bioengineered Wnts and single cell RNA-seq approaches (Yan et al., Nature, 2017a; Janda et al, Nature 2017b). We have found that Bmi1+ ISC are strongly injury-inducible versusthe homeostatic function of Lgr5+ ISC (c.f. Yan et al, PNAS 2012, Barry et al, Nature 2013). Further, we have derived robust organoid methods for prolonged culture of and ex vivo expansion of primary intestine and other GI organs, with preservation of ISCs and recapitulation of the Wnt- and Notch-dependent ISC niche, even allowing peristalsis (Ootani et al, Nat Med 2009; Li et al Nat Med 2014).
Angiogenesis and the blood-brain barrier
We are interested in determining functions of novel molecules regulating angiogenesis including receptors such as GPCRs, microRNAs and secreted molecules. We found that GPR124 is essential for developmental brain angiogenesis (Kuhnert et al, Science 2010) and that GPR124 is critical for maintaining blood-brain barrier integrity during stroke and brain tumor growth (Chang et al, Nat Med 2017). We have several active projects in stroke and blood-brain barrier (BBB) biology. We are also exploring the functions of the endothelial miRNA miR-126 in adults using conditional ko mice (Kuhnert et al, Development 2008). We have extensive experience using adenoviral expression of soluble receptor ectodomains to inhibit diverse angiogenic pathways including VEGF and PDGFRb. Loss-of-function phenotypes would simulate the effects of pharmacologic inhibition of novel targets for anti-angiogenic therapy of cancer and ocular disorders.
Endothelial cell regulation of physiology,
How do endothelial cells regulate physiology of their host organs? The liver hepatocyte appears particularly responsive to its host endothelial cells. We are investigating effects of VEGF inhibition on hepatocyte functions in terms of Epo synthesis, erythropoiesis (Tam et al, Nat Med 2006) and metabolic pathways. We have found that FDA-approved VEGF inhibitors such as aflibercept improve glucose tolerance and can treat animal models of diabetes, by stimulating cross-talk between the hypoxia and insulin signaling pathways, with sensitization of hepatic insulin signaling (Wei et al., Nature Med 2013a; Taniguchi et al., Nat. Med., 2013b). We are also correlating these changes with anti-tumor response and survival in cancer patients receiving VEGF inhibitors, as potential surrogate biomarkers of efficacy.
Independent Studies (17)
- Directed Investigation
BIOE 392 (Win)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Teaching in Immunology
IMMUNOL 290 (Aut, Sum)
- Undergraduate Research
IMMUNOL 199 (Aut, Sum)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Directed Investigation
intestinal stem-cell self-renewal.
2017; 545 (7653): 238-242
The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5(+) intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5(+) ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5(+) ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5(+) ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.
View details for DOI 10.1038/nature22313
View details for PubMedID 28467820
Surrogate Wnt agonists that phenocopy canonical Wnt and beta-catenin signalling
2017; 545 (7653): 234-?
Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.
View details for DOI 10.1038/nature22306
View details for Web of Science ID 000400963800036
View details for PubMedID 28467818
Gpr124 is essential for blood-brain barrier integrity in central nervous system disease
2017; 23 (4): 450-?
Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.
View details for DOI 10.1038/nm.4309
View details for Web of Science ID 000398768100013
View details for PubMedID 28288111
Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states
2017; 23 (2): 174-184
Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N(4)-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.
View details for DOI 10.1038/nm.4267
View details for Web of Science ID 000393729000009
View details for PubMedID 28092664
- Toward recreating colon cancer in human organoids. Nature medicine 2015; 21 (3): 215-216
Ascl2 reinforces intestinal stem cell identity.
Cell stem cell
2015; 16 (2): 105-106
Ascl2 is a Wnt-responsive master transcription factor that controls the Lgr5(+) intestinal stem cell gene expression program. Now in Cell Stem Cell, Schuijers et al. (2015) report an Ascl2 positive feedback loop, tuned by previous Wnt pathway activity, that perpetuates intestinal stem cell identity in response to Wnt/R-spondin stimulation.
View details for DOI 10.1016/j.stem.2015.01.014
View details for PubMedID 25658363
Identification and specification of the mouse skeletal stem cell.
2015; 160 (1-2): 285-298
How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.
View details for DOI 10.1016/j.cell.2014.12.002
View details for PubMedID 25594184
View details for PubMedCentralID PMC4297645
- Through-skull fluorescence imaging of the brain in a new near-infrared window NATURE PHOTONICS 2014; 8 (9): 723-730
Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture
2014; 20 (7): 769-777
The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.
View details for DOI 10.1038/nm.3585
View details for Web of Science ID 000338689500021
Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer.
2014; 15 (8): 428-?
Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.
View details for DOI 10.1186/s13059-014-0428-9
View details for PubMedID 25315765
View details for PubMedCentralID PMC4145231
Interfollicular Epidermal Stem Cells Self-Renew via Autocrine Wnt Signaling
2013; 342 (6163): 1226-1230
The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.
View details for DOI 10.1126/science.1239730
View details for Web of Science ID 000327857900046
View details for PubMedID 24311688
A liver Hif-2a-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition.
2013; 19 (10): 1331-1337
Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.
View details for DOI 10.1038/nm.3295
View details for PubMedID 24037094
- A liver Hif-2 alpha-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition NATURE MEDICINE 2013; 19 (10): 1331-?
Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes.
2013; 19 (10): 1325-1330
Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain-containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes. Although Hif regulates many metabolic pathways, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.
View details for DOI 10.1038/nm.3294
View details for PubMedID 24037093
View details for PubMedCentralID PMC4089950
Restriction of intestinal stem cell expansion and the regenerative response by YAP
2013; 493 (7430): 106-?
A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.
View details for DOI 10.1038/nature11693
View details for Web of Science ID 000312933800040
View details for PubMedID 23178811
beta-Catenin-Driven Cancers Require a YAP1 Transcriptional Complex for Survival and Tumorigenesis
2012; 151 (7): 1457-1473
Wnt/β-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of β-catenin in transformation, we classified β-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that β-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with β-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of β-catenin-dependent cancers in both cell lines and animal models. These observations define a β-catenin-YAP1-TBX5 complex essential to the transformation and survival of β-catenin-driven cancers.
View details for DOI 10.1016/j.cell.2012.11.026
View details for Web of Science ID 000312890300012
View details for PubMedID 23245941
The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (2): 466-471
The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.
View details for DOI 10.1073/pnas.1118857109
View details for Web of Science ID 000298950200030
View details for PubMedID 22190486
View details for PubMedCentralID PMC3258636
Essential Regulation of CNS Angiogenesis by the Orphan G Protein-Coupled Receptor GPR124
2010; 330 (6006): 985-989
The orphan G protein-coupled receptor (GPCR) GPR124/tumor endothelial marker 5 is highly expressed in central nervous system (CNS) endothelium. Here, we show that complete null or endothelial-specific GPR124 deletion resulted in embryonic lethality from CNS-specific angiogenesis arrest in forebrain and neural tube. Conversely, GPR124 overexpression throughout all adult vascular beds produced CNS-specific hyperproliferative vascular malformations. In vivo, GPR124 functioned cell-autonomously in endothelium to regulate sprouting, migration, and developmental expression of the blood-brain barrier marker Glut1, whereas in vitro, GPR124 mediated Cdc42-dependent directional migration to forebrain-derived, vascular endothelial growth factor-independent cues. Our results demonstrate CNS-specific angiogenesis regulation by an endothelial receptor and illuminate functions of the poorly understood adhesion GPCR subfamily. Further, the functional tropism of GPR124 marks this receptor as a therapeutic target for CNS-related vascular pathologies.
View details for DOI 10.1126/science.1196554
View details for Web of Science ID 000284118000049
View details for PubMedID 21071672
Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.
2009; 15 (6): 701-706
The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.
View details for DOI 10.1038/nm.1951
View details for PubMedID 19398967
Wnt pathway regulation of intestinal stem cells.
journal of physiology
2016; 594 (17): 4837-4847
Wnt signalling is involved in multiple aspects of embryonic development and adult tissue homeostasis, notably via controlling cellular proliferation and differentiation. Wnt signalling is subject to stringent positive and negative regulation to promote proper development and homeostasis yet avoid aberrant growth. Such multi-layer regulation includes post-translational modification and processing of Wnt proteins themselves, R-spondin (Rspo) amplification of Wnt signalling, diverse receptor families, and intracellular and extracellular antagonists and destruction and transcription complexes. In the gastrointestinal tract, Wnt signalling is crucial for development and renewal of the intestinal epithelium. Intestinal stem cells (ISCs) undergo symmetric division and neutral drift dynamics to renew the intestinal epithelium. Sources of Wnts and Wnt amplifers such as R-spondins are beginning to be elucidated as well as their functional contribution to intestinal homeostasis. In this review we focus on regulation of ISCs and intestinal homeostasis by the Wnt/Rspo pathway, the potential cellular sources of Wnt signalling regulators and highlight potential future areas of study.
View details for DOI 10.1113/JP271754
View details for PubMedID 27581568
- Transforming Big Data into Cancer-Relevant Insight: An Initial, Multi-Tier Approach to Assess Reproducibility and Relevance The Cancer Target Discovery and Development Network MOLECULAR CANCER RESEARCH 2016; 14 (8): 675-682
Kruppel-like Factor 4 Modulates Development of BMI1(+) Intestinal Stem Cell-Derived Lineage Following gamma-Radiation-Induced Gut Injury in Mice
STEM CELL REPORTS
2016; 6 (6): 815-824
In response to ionizing radiation-induced injury, the normally quiescent intestinal stem cells marked by BMI1 participate in the regenerative response. Previously, we established a protective role for Krüppel-like factor 4 (KLF4) in the intestinal epithelium where it reduces senescence, apoptosis, and crypt atrophy following γ-radiation-induced gut injury. We also described a pro-proliferative function for KLF4 during the regenerative phase post irradiation. In the current study, using a mouse model in which Klf4 is deleted from quiescent BMI1(+) intestinal stem cells, we observed increased proliferation from the BMI1(+) lineage during homeostasis. In contrast, following irradiation, Bmi1-specific Klf4 deletion leads to decreased expansion of the BMI1(+) lineage due to a combination of reduced proliferation and increased apoptosis. Our results support a critical role for KLF4 in modulating BMI1(+) intestinal stem cell fate in both homeostasis and the regenerative response to radiation injury.
View details for DOI 10.1016/j.stemcr.2016.04.014
View details for Web of Science ID 000378032600005
View details for PubMedID 27237377
Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis
2016; 35 (9): 924-941
Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.
View details for Web of Science ID 000375681600005
View details for PubMedID 26856890
View details for PubMedCentralID PMC5207321
The Wnt7's Tale: A story of an orphan who finds her tie to a famous family
2016; 107 (5): 576-582
The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008-2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood-brain barrier formation.
View details for DOI 10.1111/cas.12924
View details for Web of Science ID 000378714600002
View details for PubMedID 26934061
An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues.
Methods in molecular biology (Clifton, N.J.)
2016; 1422: 33-40
Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.
View details for DOI 10.1007/978-1-4939-3603-8_4
View details for PubMedID 27246020
Organoids as Models for Neoplastic Transformation
ANNUAL REVIEW OF PATHOLOGY: MECHANISMS OF DISEASE, VOL 11
2016; 11: 199-220
Cancer models strive to recapitulate the incredible diversity inherent in human tumors. A key challenge in accurate tumor modeling lies in capturing the panoply of homo- and heterotypic cellular interactions within the context of a three-dimensional tissue microenvironment. To address this challenge, researchers have developed organotypic cancer models (organoids) that combine the 3D architecture of in vivo tissues with the experimental facility of 2D cell lines. Here we address the benefits and drawbacks of these systems, as well as their most recent advances. In particular, we focus on the application of such models to the discovery of novel cancer drivers, the study of tumor biology, and the development of novel therapeutic approaches for the treatment of cancer. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease Volume 11 is May 23, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
View details for DOI 10.1146/annurev-pathol-012615-044249
View details for Web of Science ID 000377037200009
View details for PubMedID 26907527
- Novel TIA biomarkers identified by mass spectrometry-based proteomics INTERNATIONAL JOURNAL OF STROKE 2015; 10 (8): 1204-1211
- Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 2015; 54 (49): 14758-14762
Chemodetection and Destruction of Host Urea Allows Helicobacter pylori to Locate the Epithelium
CELL HOST & MICROBE
2015; 18 (2): 147-156
The gastric pathogen Helicobacter pylori interacts intimately with the gastric mucosa to avoid the microbicidal acid in the stomach lumen. The cues H. pylori senses to locate and colonize the gastric epithelium have not been well defined. We show that metabolites emanating from human gastric organoids rapidly attract H. pylori. This response is largely controlled by the bacterial chemoreceptor TlpB, and the main attractant emanating from epithelia is urea. Our previous structural analyses show that TlpB binds urea with high affinity. Here we demonstrate that this tight binding controls highly sensitive responses, allowing detection of urea concentrations as low as 50 nM. Attraction to urea requires that H. pylori urease simultaneously destroys the signal. We propose that H. pylori has evolved a sensitive urea chemodetection and destruction system that allows the bacterium to dynamically and locally modify the host environment to locate the epithelium.
View details for DOI 10.1016/j.chom.2015.07.002
View details for Web of Science ID 000359601800007
View details for PubMedID 26269952
- Oligodendrocyte precursors migrate along vasculature in the developing nervous system SCIENCE 2015; 351 (6271): 379-384
- Organoid modeling for cancer precision medicine. Genome medicine 2015; 7 (1): 32-?
Developmental and pathological angiogenesis in the central nervous system.
Cellular and molecular life sciences
2014; 71 (18): 3489-3506
Angiogenesis, the formation of new blood vessels from pre-existing vessels, in the central nervous system (CNS) is seen both as a normal physiological response as well as a pathological step in disease progression. Formation of the blood-brain barrier (BBB) is an essential step in physiological CNS angiogenesis. The BBB is regulated by a neurovascular unit (NVU) consisting of endothelial and perivascular cells as well as vascular astrocytes. The NVU plays a critical role in preventing entry of neurotoxic substances and regulation of blood flow in the CNS. In recent years, research on numerous acquired and hereditary disorders of the CNS has increasingly emphasized the role of angiogenesis in disease pathophysiology. Here, we discuss molecular mechanisms of CNS angiogenesis during embryogenesis as well as various pathological states including brain tumor formation, ischemic stroke, arteriovenous malformations, and neurodegenerative diseases.
View details for DOI 10.1007/s00018-014-1625-0
View details for PubMedID 24760128
Engineering of three-dimensional microenvironments to promote contractile behavior in primary intestinal organoids.
2014; 6 (2): 127-142
Multiple culture techniques now exist for the long-term maintenance of neonatal primary murine intestinal organoids in vitro; however, the achievement of contractile behavior within cultured organoids has thus far been infrequent and unpredictable. Here we combine finite element simulation of oxygen transport and quantitative comparative analysis of cellular microenvironments to elucidate the critical variables that promote reproducible intestinal organoid contraction. Experimentally, oxygen distribution was manipulated by adjusting the ambient oxygen concentration along with the use of semi-permeable membranes to enhance transport. The culture microenvironment was further tailored through variation of collagen type-I matrix density, addition of exogenous R-spondin1, and specification of culture geometry. "Air-liquid interface" cultures resulted in significantly higher numbers of contractile cultures relative to traditional submerged cultures. These interface cultures were confirmed to have enhanced and more symmetric oxygen transport relative to traditional submerged cultures. While oxygen availability was found to impact in vitro contraction rate and the orientation of contractile movement, it was not a key factor in enabling contractility. For all conditions tested, reproducible contractile behavior only occurred within a consistent and narrow range of collagen type-I matrix densities with porosities of approximately 20% and storage moduli near 30 Pa. This suggests that matrix density acts as a "permissive switch" that enables contractions to occur. Similarly, contractions were only observed in cultures with diameters less than 15.5 mm that had relatively large interfacial surface area between the compliant matrix and the rigid culture dish. Taken together, these data suggest that spatial geometry and mechanics of the microenvironment, which includes both the encapsulating matrix as well as the surrounding culture device, may be key determinants of intestinal organoid functionality. As peristaltic contractility is a crucial requirement for normal digestive tract function, this achievement of reproducible organoid contraction marks a pivotal advancement towards engineering physiologically functional replacement tissue constructs.
View details for DOI 10.1039/c3ib40188j
View details for PubMedID 24343706
Partial Proteasome Inhibitors Induce Hair Follicle Growth by Stabilizing ß-Catenin.
2014; 32 (1): 85-92
The activation of tissue stem cells from their quiescent state represents the initial step in the complex process of organ regeneration and tissue repair. While the identity and location of tissue stem cells are becoming known, how key regulators control the balance of activation and quiescence remains mysterious. The vertebrate hair is an ideal model system where hair cycling between growth and resting phases is precisely regulated by morphogen signaling pathways, but how these events are coordinated to promote orderly signaling in a spatial and temporal manner remains unclear. Here, we show that hair cycle timing depends on regulated stability of signaling substrates by the ubiquitin-proteasome system. Topical application of partial proteasomal inhibitors (PaPIs) inhibits epidermal and dermal proteasome activity throughout the hair cycle. PaPIs prevent the destruction of the key anagen signal β-catenin, resulting in more rapid hair growth and dramatically shortened telogen. We show that PaPIs induce excess β-catenin, act similarly to the GSK3β antagonist LiCl, and antagonize Dickopf-related protein-mediated inhibition of anagen. PaPIs thus represent a novel class of hair growth agents that act through transiently modifying the balance of stem cell activation and quiescence pathways. Stem Cells 2014;32:85-92.
View details for DOI 10.1002/stem.1525
View details for PubMedID 23963711
- Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer GENOME BIOLOGY 2014; 15 (8)
A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2013; 305 (8): G542-G551
Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM(+)/CD44(+)) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
View details for DOI 10.1152/ajpgi.00481.2012
View details for Web of Science ID 000325809200002
View details for PubMedID 23928185
A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
2013; 305 (8): G542-G551
Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
View details for DOI 10.1152/ajpgi.00481.2012
View details for Web of Science ID 000325809200002
View details for PubMedID 23928185
View details for PubMedCentralID PMC3798732
The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO
2012; 149 (1): 63-74
Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.
View details for DOI 10.1016/j.cell.2012.01.051
View details for Web of Science ID 000302235400010
View details for PubMedID 22464323
View details for PubMedCentralID PMC3408231
- PDGF-B exploits stromal EPO NATURE MEDICINE 2012; 18 (1): 22-24
- Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling NATURE MEDICINE 2012; 18 (1): 111-119
Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling.
2012; 18 (1): 111-119
Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes--differentiated epithelial cells crucial for filtration--are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt-β-catenin pathways in podocyte proliferation and disease.
View details for DOI 10.1038/nm.2550
View details for PubMedID 22138751
Endochondral ossification is required for haematopoietic stem-cell niche formation
2009; 457 (7228): 490-U9
Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.
View details for DOI 10.1038/nature07547
View details for Web of Science ID 000262519200049
View details for PubMedID 19078959
View details for PubMedCentralID PMC2648141
Attribution of vascular phenotypes of the murine Egfl7 locus to the microRNA miR-126
2008; 135 (24): 3989-3993
Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7(Delta) and miR-126(Delta) alleles revealed that Egfl7(Delta/Delta) mice were phenotypically normal, whereas miR-126(Delta/Delta) mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85beta subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.
View details for DOI 10.1242/dev.029736
View details for Web of Science ID 000261151000002
View details for PubMedID 18987025
Soluble receptor-mediated selective inhibition of VEGFR and PDGFR beta signaling during physiologic and tumor angiogenesis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (29): 10185-10190
The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.
View details for DOI 10.1073/pnas.0803194105
View details for Web of Science ID 000257913200061
View details for PubMedID 18632559
Recombinant adenovirus as a methodology for exploration of physiologic functions of growth factor pathways
JOURNAL OF MOLECULAR MEDICINE-JMM
2008; 86 (2): 161-169
The use of recombinant adenoviruses (Ad) to express secreted antagonists of growth factors represents a powerful strategy for studying physiologic functions of growth factor pathways in experimental animals. Indeed, a single adenoviral injection can produce characteristic high-level and persistent plasma expression of soluble receptor ectodomains or secreted protein antagonists, allowing highly stringent conditional inactivation of target pathways in vivo. In this review, we describe our experience using recombinant Ad to inactivate growth factor pathways in vivo and discuss their advantages and limitations. Using our studies on vascular endothelial growth factor and Wnt systems as examples, we further describe how recombinant Ad can unveil previously unknown physiological roles of signaling pathways. Finally, we discuss the potential physiological and therapeutic relevance of our findings.
View details for DOI 10.1007/s00109-007-0261-7
View details for Web of Science ID 000252799300004
View details for PubMedID 17891365
Augmented Wnt signaling in a mammalian model of accelerated aging
2007; 317 (5839): 803-806
The contribution of stem and progenitor cell dysfunction and depletion in normal aging remains incompletely understood. We explored this concept in the Klotho mouse model of accelerated aging. Analysis of various tissues and organs from young Klotho mice revealed a decrease in stem cell number and an increase in progenitor cell senescence. Because klotho is a secreted protein, we postulated that klotho might interact with other soluble mediators of stem cells. We found that klotho bound to various Wnt family members. In a cell culture model, the Wnt-klotho interaction resulted in the suppression of Wnt biological activity. Tissues and organs from klotho-deficient animals showed evidence of increased Wnt signaling, and ectopic expression of klotho antagonized the activity of endogenous and exogenous Wnt. Both in vitro and in vivo, continuous Wnt exposure triggered accelerated cellular senescence. Thus, klotho appears to be a secreted Wnt antagonist and Wnt proteins have an unexpected role in mammalian aging.
View details for DOI 10.1126/science.1143578
View details for Web of Science ID 000248624500040
View details for PubMedID 17690294
Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis
2007; 317 (5839): 807-810
The regenerative potential of skeletal muscle declines with age, and this impairment is associated with an increase in tissue fibrosis. We show that muscle stem cells (satellite cells) from aged mice tend to convert from a myogenic to a fibrogenic lineage as they begin to proliferate and that this conversion is mediated by factors in the systemic environment of the old animals. We also show that this lineage conversion is associated with an activation of the canonical Wnt signaling pathway in aged myogenic progenitors and can be suppressed by Wnt inhibitors. Furthermore, components of serum from aged mice that bind to the Frizzled family of proteins, which are Wnt receptors, may account for the elevated Wnt signaling in aged cells. These results indicate that the Wnt signaling pathway may play a critical role in tissue-specific stem cell aging and an increase in tissue fibrosis with age.
View details for DOI 10.1126/science.1144090
View details for Web of Science ID 000248624500041
View details for PubMedID 17690295
VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis
2006; 12 (7): 793-800
Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.
View details for DOI 10.1038/nm1428
View details for Web of Science ID 000238862800066
View details for PubMedID 16799557
Apc tumor suppressor gene is the "zonation-keeper" of mouse liver
2006; 10 (6): 759-770
The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.
View details for DOI 10.1016/j.devcel.2006.03.015
View details for Web of Science ID 000238244700010
View details for PubMedID 16740478
Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (1): 266-271
Whereas the adult gastrointestinal epithelium undergoes tremendous self-renewal through active proliferation in crypt stem cell compartments, the responsible growth factors regulating this continuous proliferation have not been defined. The exploration of physiologic functions of Wnt proteins in adult organisms has been hampered by functional redundancy and the necessity for conditional inactivation strategies. Dickkopf-1 (Dkk1) is a potent secreted Wnt antagonist that interacts with Wnt coreceptors of the LRP family. To address the contribution of Wnt signaling to gastrointestinal epithelial proliferation, adenoviral expression of Dkk1 was used to achieve stringent, conditional, and reversible Wnt inhibition in adult animals. Adenovirus Dkk1 (Ad Dkk1) treatment of adult mice repressed expression of the Wnt target genes CD44 and EphB2 within 2 days in both small intestine and colon, indicating an extremely broad role for Wnt signaling in the maintenance of adult gastrointestinal gene expression. In parallel, Ad Dkk1 markedly inhibited proliferation in small intestine and colon, accompanied by progressive architectural degeneration with the loss of crypts, villi, and glandular structure by 7 days. Whereas decreased Dkk1 expression at later time points (>10 days) was followed by crypt and villus regeneration, which was consistent with a reversible process, substantial mortality ensued from colitis and systemic infection. These results indicate the efficacy of systemic expression of secreted Wnt antagonists as a general strategy for conditional inactivation of Wnt signaling in adult organisms and illustrate a striking reliance on a single growth factor pathway for the maintenance of the architecture of the adult small intestine and colon.
View details for Web of Science ID 000187937200050
View details for PubMedID 14695885
View details for PubMedCentralID PMC314174
Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2001; 98 (8): 4605-4610
Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in approximately 80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.
View details for Web of Science ID 000168059700062
View details for PubMedID 11274374
Antiangiogenic gene therapy using soluble VEGF receptors.
AMER SOC HEMATOLOGY. 2000: 211A–211A
View details for Web of Science ID 000165256100903
RAPAMYCIN SELECTIVELY INHIBITS INTERLEUKIN-2 ACTIVATION OF P70 S6 KINASE
1992; 358 (6381): 70-73
The macrolide rapamycin induces cell cycle G1 arrest in yeast and in mammalian cells, which suggests that an evolutionarily conserved, rapamycin-sensitive pathway may regulate entry into S phase. In mammals, rapamycin inhibits interleukin-2 receptor-induced S phase entry and subsequent T-cell proliferation, resulting in immunosuppression. Here we show that interleukin-2 selectively stimulates the phosphorylation and activation of p70 S6 kinase but not the erk-encoded MAP kinases and rsk-encoded S6 kinases. Rapamycin completely and rapidly inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase at concentrations comparable to those blocking S phase entry of T cells (0.05-0.2 nM). The structurally related macrolide FK506 competitively antagonizes the actions of rapamycin, indicating that these effects are mediated by FKBP, which binds the transition-state mimic structure common to both rapamycin and FK506 (refs 4, 6, 9-11). The selective blockade of the p70 S6 kinase activation cascade by the rapamycin-FKBP complex implicates this signalling pathway in the regulation of T cell entry into S phase.
View details for Web of Science ID A1992JB34100056
View details for PubMedID 1614535
A TRANSCRIPTIONAL HIERARCHY INVOLVED IN MAMMALIAN CELL-TYPE SPECIFICATION
1992; 355 (6359): 457-461
Although transcriptional hierarchies have been extensively studied in invertebrates, their involvement in mammalian cell-type specification is poorly understood. Here we report a hepatocyte transcriptional cascade suggested by the expression patterns of hepatic transcription factors in dedifferentiated hepatomas and hepatocyte: fibroblast hybrids in which the liver phenotype was extinguished. These results indicated that the homeoprotein hepatocyte nuclear factor-1 alpha (HNF-1 alpha), and HNF-4, a member of the steroid hormone receptor superfamily, were regulated coordinately or in a hierarchy by a higher-order locus, independently of other hepatic transactivators. HNF-4 was implicated as an essential positive regulator of HNF-1 alpha, as deletion of an HNF-4 binding site in the HNF-1 alpha promoter abolished promoter activity, and HNF-4 potently transactivated the HNF-1 alpha promoter in cotransfection assays. Moreover, genetic complementation of dedifferentiated hepatomas with HNF-4 complementary DNA rescued expression of endogenous HNF-1 alpha messenger RNA and DNA-binding activity. Our studies therefore define an HNF-4----HNF-1 alpha (4----1 alpha) transcriptional hierarchy operative in differentiated hepatocytes but selectively inhibited by an extinguishing locus and somatic mutations which antagonize the liver phenotype.
View details for Web of Science ID A1992HB53000073
View details for PubMedID 1734282