Telomere maintenance is essential for the continuous growth of tumor cells. In most human tumors telomeres are maintained by telomerase, a specialized reverse transcriptase. Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase (PARP), positively regulates telomere length through its interaction with TRF1, a telomeric DNA-binding protein. Tankyrase 1 ADP-ribosylates TRF1, inhibiting its binding to telomeric DNA. Overexpression of tankyrase 1 in the nucleus promotes telomere elongation, suggesting that tankyrase 1 regulates access of telomerase to the telomeric complex. The recent identification of a closely related homolog of tankyrase 1, tankyrase 2, opens the possibility for a second PARP at telomeres. We therefore sought to establish the role of tankyrase 1 at telomeres and to determine if tankyrase 2 might have a telomeric function. We show that endogenous tankyrase 1 is a component of the human telomeric complex. We demonstrate that telomere elongation by tankyrase 1 requires the catalytic activity of the PARP domain and does not occur in telomerase-negative primary human cells. To investigate a potential role for tankyrase 2 at telomeres, recombinant tankyrase 2 was subjected to an in vitro PARP assay. Tankyrase 2 poly(ADP-ribosyl)ated itself and TRF1. Overexpression of tankyrase 2 in the nucleus released endogenous TRF1 from telomeres. These findings establish tankyrase 2 as a bona fide PARP, with itself and TRF1 as acceptors of ADP-ribosylation, and suggest the possibility of a role for tankyrase 2 at telomeres.Telomere integrity is essential for chromosome stability, and the maintenance of telomeric DNA is required for long-term proliferation of eukaryotic cells. Telomeres are maintained by telomerase, a reverse transcriptase that adds telomeric repeats to chromosome ends (14; reviewed in reference 31). In most normal human somatic tissue telomerase is repressed, and as a result, telomeres shorten (17, 18). Critically short telomeres lose their ability to protect chromosome ends, resulting in chromosomal degradation and fusion. In contrast to normal somatic human cells, immortalized cells (including cancer cells) and germ cells express telomerase (21, 34) and maintain their telomeres. In these cells telomere maintenance is regulated by a homeostatic mechanism (reviewed in reference 29). Thus, in the mammalian germ line telomeres show a speciesspecific telomere length setting which is constant over the generations (23). Regulation is also apparent in many human tumor cell lines, where despite the presence of high levels of telomerase telomeres do not grow, but rather, they are stably maintained within a given size range (9, 10).Mammalian telomeres consist of long tandem arrays of TTAGGG repeats bound by the DNA-binding proteins, TRF1 and TRF2 (4, 5, 7; reviewed in reference 8). The TRFs are related in their primary structure; both contain carboxy-terminal Myb-type DNA-binding motifs and internal, conserved domains required for homodimerization (2, 5). The proteins do not form heterodimers (5). A dis...

VEGF and TGF‐β1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF‐β1 induces apoptosis. We have previously shown that VEGF/VEGF receptor‐2 (VEGFR2) signaling mediates TGF‐β1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF‐mediated apoptosis is required for TGF‐β1 induction of angiogenesis. In vitro the apoptotic effect of TGF‐β1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF‐β1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord‐like structures by TGF‐β1 with an effect comparable to that of z‐VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF‐β1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF‐β1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF‐β1 induction of both apoptosis and angiogenesis, an effect similar to that of z‐VAD. Thus, TGF‐β1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF‐mediated apoptosis as a potential target to control angiogenesis. J. Cell. Physiol. 219: 449–458, 2009. © 2009 Wiley‐Liss, Inc.

VEGF and TGF-β1 induce angiogenesis but have opposing effects on endothelial cells.

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardation phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement for Tnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together, these results suggest that Tnks2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.

Diabetes is linked to loss of pancreatic beta-cells. Pluripotent stem cells offer a valuable source of human beta-cells for basic studies of their biology and translational applications. However, the signalling pathways that regulate beta-cell development and functional maturation are not fully understood. Here we report a high content chemical screen, revealing that H1152, a ROCK inhibitor, promotes the robust generation of insulin-expressing cells from multiple hPSC lines. The insulin expressing cells obtained after H1152 treatment show increased expression of mature beta cell markers and improved glucose stimulated insulin secretion. Moreover, the H1152-treated beta-like cells show enhanced glucose stimulated insulin secretion and increased capacity to maintain glucose homeostasis after transplantation. Conditional gene knockdown reveals that inhibition of ROCKII promotes the generation and maturation of glucose-responding cells. This study provides a strategy to promote human beta-cell maturation and identifies an unexpected role for the ROCKII pathway in the development and maturation of beta-like cells.

GLIS3 mutations are associated with type 1, type 2, and neonatal diabetes, reflecting a key function for this gene in pancreatic β-cell biology. Previous attempts to recapitulate disease-relevant phenotypes in GLIS3−/− β-like cells have been unsuccessful. Here, we develop a “minimal component” protocol to generate late-stage pancreatic progenitors (PP2) that differentiate to mono-hormonal glucose-responding β-like (PP2-β) cells. Using this differentiation platform, we discover that GLIS3−/− hESCs show impaired differentiation, with significant death of PP2 and PP2-β cells, without impacting the total endocrine pool. Furthermore, we perform a high-content chemical screen and identify a drug candidate that rescues mutant GLIS3-associated β-cell death both in vitro and in vivo. Finally, we discovered that loss of GLIS3 causes β-cell death, by activating the TGFβ pathway. This study establishes an optimized directed differentiation protocol for modeling human β-cell disease and identifies a drug candidate for treating a broad range of GLIS3-associated diabetic patients.

Summary Sorely missing from the “toolkit” for directed differentiation of stem/progenitor cells are agonists of the BMP signaling pathway. Using a high throughput chemical screen, we discovered that PD407824, a checkpoint kinase 1 (CHK1) inhibitor, increases the sensitivity of cells to sub-threshold amounts of BMP4. We show utility of the compound in the directed differentiation of human embryonic stem cells toward mesoderm or cytotrophoblast stem cells. Blocking CHK1 activity using pharmacological compounds or CHK1 knockout using sgRNA confirmed that CHK1 inhibition increases the sensitivity to BMP4 treatment. Additional mechanistic studies indicate that CHK1 inhibition depletes p21 levels, thereby activating CDK8/9, which then phosphorylates the SMAD2/3 linker region, leading to decreased levels of SMAD2/3 protein and enhanced levels of nuclear SMAD1. This study provides insight into mechanisms controlling the BMP/TGFβ signaling pathways, and a useful pharmacological reagent for directed differentiation of stem cells.

Bone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins, including SMAD1. In previous work, we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly, we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely, when Smad1 is depleted in FlK1 ؉ mesoderm, at a stage after hemangioblast commitment, the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis, including Gata1, Runx1 and Eklf, rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity, indicating molecular cross-regulation between the BMP and TGF-␤ signaling pathways. Consistent with this mechanism, hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore, this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors. (Blood. 2011; 117(24):6489-6497) IntroductionMammalian hematopoiesis begins on the yolk sac, generating a transient population of primitive erythrocytes, as well as a subset of definitive myeloid lineages. 1 Considerable progress has been made identifying the relevant signaling pathways that regulate early hematopoiesis. 2,3 The bone morphogenetic protein (BMP) members of the TGF-␤ superfamily are particularly important in patterning the character of early embryonic tissue to generate ventral mesoderm, 4,5 which serves as the origin of the developing hemangioblast, a cell type that is bipotent for the hematopoietic and vascular lineages. 6 Forced expression of BMP ligands or signaling components expands hematopoiesis, 7 while genetic deletion of Bmp4 or its cognate receptor results in developmental arrest during gastrulation, with attendant failure to generate the mesodermal precursors from the epiblast that are required for hematopoietic development. 4,8 Key downstream effectors of TGF-␤ and BMP signaling are members of the SMAD family of transcription factors, that are activated by type I receptors through C-terminal serine/threonine phosphorylation, after ligand binding and subsequent transactivation of type I/type II receptor heterodimers. 9 There are 5 receptor-activated SMADs, or R-SMADS: SMAD2 and SMAD3 mediate the TGF-␤/Activin/Nodal pathway, while SMADs 1, 5, and 8 respond to BMP signaling. Smad1 and Smad5 have roles in hematopoiesis, while Smad8 is apparently not expressed in hematopoietic cells. 10 Activated R-SMADs compete for access to a single coactivator or coSMAD, SMAD4, tha...