Ling Yan

PP2A regulates the pro-apoptotic activity of FOXO1.

FOXO1, a member of the evolutionarily conserved forkhead family of transcription factors, regulates expression of a number of genes that play critical roles in cell cycle and apoptosis. A pivotal regulatory mechanism of FOXO is reversible phosphorylation, catalyzed by kinases and phosphatases. Phosphorylation of FOXO1 is associated with 14-3-3 binding and cytosolic localization, whereas dephosphorylated FOXO1 translocates to the nucleus and is transcriptionally active. Experiments were performed to identify the serine/threonine phosphatase that dephosphorylates FOXO1. PP2A inhibitors, okadaic acid and fostriecin, increased FOXO1 phosphorylation in vitro and in cells. Microcystin-agarose pull-downs suggested that a phosphatase binds to FOXO1, and PP2A catalytic subunit was identified in endogenous FOXO1 immunocomplexes, indicating that PP2A is a FOXO1 phosphatase. Purified PP2A interacted directly with FOXO1 and dephosphorylated FOXO1 in vitro. Silencing of PP2A protected FOXO1 from dephosphorylation and delayed FOXO1 nuclear translocation, confirming the physiologic role of PP2A in the regulation of FOXO1 function. Furthermore, inhibition of PP2A phosphatases rescued FOXO1-mediated cell death by regulating the level of the pro-apoptotic protein BIM. We conclude that PP2A is a physiologic phosphatase of FOXO1.

G0 function of BCL2 and BCL-xL requires BAX, BAK, and p27 phosphorylation by Mirk, revealing a novel role of BAX and BAK in quiescence regulation.

BCL2 and BCL-xL facilitate G0 quiescence by decreasing RNA content and cell size and up-regulating p27 protein, but the precise mechanism is not understood. We investigated the relationship between cell cycle regulation and the anti-apoptosis function of BCL2 and BCL-xL. Neither caspase inhibition nor abrogation of mitochondria-dependent apoptosis by BAX and BAK deletion fully recapitulated the G0 effects of BCL2 or BCL-xL, suggesting that mechanisms in addition to anti-apoptosis are involved in the cell cycle arrest function of BCL2 or BCL-xL. We found that BCL2 and BCL-xL expression in bax-/- bak-/- cells did not confer cell cycle effects, consistent with the G0 function of BCL2 and BCL-xL being mediated through BAX or BAK. Stabilization of p27 in G0 in BCL2 or BCL-xL cells was due to phosphorylation of p27 at Ser10 by the kinase Mirk. In bax-/- bak-/- cells, total p27 and p27 phosphorylated at Ser10 were elevated. Re-expression of BAX in bax-/- bak-/- cells and silencing of BAX and BAK in wild type cells confirmed that endogenous BAX and BAK modulated p27. These data revealed a novel role for BAX and BAK in the regulation of G0 quiescence.

Bone Marrow–derived Cells Contribute to the Pathogenesis of Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension (PAH) is a progressive lung disease of the pulmonary microvasculature. Studies suggest that bone marrow (BM)-derived circulating cells may play an important role in its pathogenesis. OBJECTIVES We used a genetic model of PAH, the Bmpr2 mutant mouse, to study the role of BM-derived circulating cells in its pathogenesis. METHODS Recipient mice, either Bmpr2(R899X) mutant or controls, were lethally irradiated and transplanted with either control or Bmpr2(R899X) BM cells. Donor cells were traced in female recipient mice by Y chromosome painting. Molecular and function insights were provided by expression and cytokine arrays combined with flow cytometry, colony-forming assays, and competitive transplant assays. MEASUREMENTS AND MAIN RESULTS We found that mutant BM cells caused PAH with remodeling and inflammation when transplanted into control mice, whereas control BM cells had a protective effect against the development of disease, when transplanted into mutant mice. Donor BM-derived cells were present in the lungs of recipient mice. Functional and molecular analysis identified mutant BM cell dysfunction suggestive of a PAH phenotype soon after activation of the transgene and long before the development of lung pathology. CONCLUSIONS Our data show that BM cells played a key role in PAH pathogenesis and that the transplanted BM cells were able to drive the lung phenotype in a myeloablative transplant model. Furthermore, the specific cell types involved were derived from hematopoietic stem cells and exhibit dysfunction long before the development of lung pathology.

Tgif1 regulates quiescence and self-renewal of hematopoietic stem cells

ABSTRACT TG-interacting factor 1 (TGIF1) is a transcriptional repressor that can modulate retinoic acid and transforming growth factor β signaling pathways. It is required for myeloid progenitor cell differentiation and survival, and mutations in the TGIF1 gene cause holoprosencephaly. Furthermore, we have previously observed that acute myelogenous leukemia (AML) patients with low TGIF1 levels had worse prognoses. Here, we explored the role of Tgif1 in murine hematopoietic stem cell (HSC) function. CFU assays showed that Tgif1−/− bone marrow cells produced more total colonies and had higher serial CFU potential. These effects were also observed in vivo, where Tgif1−/− bone marrow cells had higher repopulation potential in short- and long-term competitive repopulation assays than wild-type cells. Serial transplantation and replating studies showed that Tgif1−/− HSCs exhibited greater self-renewal and were less proliferative and more quiescent than wild-type cells, suggesting that Tgif1 is required for stem cells to enter the cell cycle. Furthermore, HSCs from Tgif1+/− mice had a phenotype similar to that of HSCs from Tgif1−/− mice, while bone marrow cells with overexpressing Tgif1 showed increased proliferation and lower survival in long-term transplant studies. Taken together, our data suggest that Tgif1 suppresses stem cell self-renewal and provide clues as to how reduced expression of TGIF1 may contribute to poor long-term survival in patients with AML.

The B55α-containing PP2A holoenzyme dephosphorylates FOXO1 in islet β-cells under oxidative stress

The FOXO1 (forkhead box O1) transcription factor influences many key cellular processes, including those important in metabolism, proliferation and cell death. Reversible phosphorylation of FOXO1 at Thr(24) and Ser(256) regulates its subcellular localization, with phosphorylation promoting cytoplasmic localization, whereas dephosphorylation triggers nuclear import and transcriptional activation. In the present study, we used biochemical and molecular approaches to isolate and link the serine/threonine PP2A (protein phosphatase 2A) holoenzyme containing the B55α regulatory subunit, with nuclear import of FOXO1 in pancreatic islet β-cells under oxidative stress, a condition associated with cellular dysfunction in Type 2 diabetes. The mechanism of FOXO1 dephosphorylation and nuclear translocation was investigated in pancreatic islet INS-1 and βTC-3 cell lines subjected to oxidative stress. A combined chemical cross-linking and MS strategy revealed the association of FOXO1 with a PP2A holoenzyme composed of the catalytic C, structural A and B55α regulatory subunits. Knockdown of B55α in INS-1 cells reduced FOXO1 dephosphorylation, inhibited FOXO1 nuclear translocation and attenuated oxidative stress-induced cell death. Furthermore, both B55α and nuclear FOXO1 levels were increased under hyperglycaemic conditions in db/db mouse islets, an animal model of type 2 diabetes. We conclude that B55α-containing PP2A is a key regulator of FOXO1 activity in vivo.

Phosphatases and regulation of cell death.

Reversible phosphorylation, catalyzed by kinases and phosphatases, regulates many apoptosis molecules. Phosphorylation inactivates, whereas dephosphorylation activates, the pro-apoptotic functions of select apoptotic regulators, including the BCL2 family, such as BAD, and transcription factors such as FOXOs. The apoptotic function of the BH3 molecule BAD is exquisitely regulated by phosphorylation. Although phosphorylated BAD is sequestered in the cytosol, dephosphorylated BAD translocates to the mitochondria and inactivates BCL-xL and BCL2. Analogously, Akt-phosphorylated FOXO1 is cytosolic and inactive as a transcription factor, but dephosphorylated FOXO1 translocates to the nucleus, where it regulates the expression of pro-apoptotic Bim and cell cycle inhibitors. By use of inhibitor experiments and a combination of immunoprecipitations and tagged pull-downs in interaction studies, we identified PP2A enzymes as BAD and FOXO1 phosphatases. PP2A dephosphorylation of BAD is regulated by competitive interaction of 14-3-3, PP2A, and BAD. On survival factor deprivation, PP2A dephosphorylation of pSer112 plays the gatekeeper role for subsequent dephosphorylation at pSer136 and pSer155 by multiple phosphatases. In contrast, PP2A and 14-3-3 can interact with FOXO1 concomitantly, but PP2A dephosphorylates the pThr24 and pSer256 only once 14-3-3 dissociates. Functional assays of cell death, Bim upregulation by FOXO1, and FOXO1 nuclear translocation in the presence of phosphatase inhibitors and phosphatase siRNAs revealed the physiologic significance of PP2A activity on BAD and FOXO1. Demonstrating the role of PP2A in regulating the function of two very different cell death molecules, a BH3 protein and a transcription factor, suggests that activation of pro-apoptotic factors by protein phosphatases may be a general regulatory mechanism in apoptosis.