John L. Cleveland

Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis

Phagocytosis and autophagy are two ancient, highly conserved processes involved, respectively, in the removal of extracellular organisms and the destruction of organisms in the cytosol. Autophagy, for either metabolic regulation or defence, involves the formation of a double membrane called the autophagosome, which then fuses with lysosomes to degrade the contents, a process that has similarities with phagosome maturation. Toll-like-receptor (TLR) engagement activates a variety of defence mechanisms within phagocytes, including facilitation of phagosome maturation, and also engages autophagy. Therefore we speculated that TLR signalling might link these processes to enhance the function of conventional phagosomes. Here we show that a particle that engages TLRs on a murine macrophage while it is phagocytosed triggers the autophagosome marker LC3 to be rapidly recruited to the phagosome in a manner that depends on the autophagy pathway proteins ATG5 and ATG7; this process is preceded by recruitment of beclin 1 and phosphoinositide-3-OH kinase activity. Translocation of beclin 1 and LC3 to the phagosome was not associated with observable double-membrane structures characteristic of conventional autophagosomes, but was associated with phagosome fusion with lysosomes, leading to rapid acidification and enhanced killing of the ingested organism.

Puma is an essential mediator of p53-dependent and -independent apoptotic pathways.

Puma encodes a BH3-only protein that is induced by the p53 tumor suppressor and other apoptotic stimuli. To assess its physiological role in apoptosis, we generated Puma knockout mice by gene targeting. Here we report that Puma is essential for hematopoietic cell death triggered by ionizing radiation (IR), deregulated c-Myc expression, and cytokine withdrawal. Puma is also required for IR-induced death throughout the developing nervous system and accounts for nearly all of the apoptotic activity attributed to p53 under these conditions. These findings establish Puma as a principal mediator of cell death in response to diverse apoptotic signals, implicating Puma as a likely tumor suppressor.

NIX is required for programmed mitochondrial clearance during reticulocyte maturation

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-XL, BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX- and BAK-independent role for a BCL2-related protein in development.

Phospholipase Cγ2 Is Essential in the Functions of B Cell and Several Fc Receptors

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

Myc pathways provoking cell suicide and cancer.

A paradox for the cancer biology field has been the revelation that oncogenes, once thought to simply provide advantages to a cancer cell, actually put it at dire risk of cell suicide. Myc is the quintessential oncogene in this respect, as in normal cells it is required for cell cycle traverse, whereas in cancers it is overexpressed and functions as the angiogenic switch. Nonetheless, Myc overexpression kills normal cells dead in their tracks. Here we review Myc-induced pathways that contribute to the apoptotic response. Molecular analysis of Myc-induced tumors has established that some of these apoptotic pathways are essential checkpoints that guard the cell from cancer, as they are selectively bypassed during tumorigenesis. The precise mechanism(s) by which Myc targets these pathways are largely unresolved, but we propose that they involve crosstalk and feedback regulatory loops between arbiters of cell death.

Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants.

The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis. Here we report that two common mutant MYC alleles derived from human Burkitts lymphoma uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the B cell lymphoma 2 (Bcl2) family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis.

Contenders in FasL/TNF death signaling

A variety of biological functions are regulated through extracellular signals. One of the most unexpected responses to emerge has been receptor-mediated control of programmed cell death, or apoptosis. Apoptosis is a morphologically defined process, characterized by the condensation of the nucleus and cytoplasm and a distinctive pattern of chromosomal DNA fragmentation. Extracellular regulation of cell death is either protective or inductive. Most growth factors initiate signaling pathways that protect cells from apoptosis. This is particularly evident in hematopoietic cells in which removal of growth factors is associated with the initiation of the cell death program. The signaling pathways that are involved in this type of regulation are not known. However, recent studies have implicated the Ras pathway (Kinoshita et al., 1995) phosphatidylinositol3-kinase (Yao and Cooper, 1995) or both. The targets of these pathways are likely to involve regulation of the Bcl-2 family of proteins, which function to suppress or activate this cell death program (Oltvai and Korsmeyer, 1994). In contrast with growth factors, Fas ligand (FasL) and the tumor necrosis factors (TNFs) rapidly and dominantly induce cell death (Nagata and Golstein, 1995). FasLinduced cell death is required for the normal elimination of potentially autoreactive peripherial T cells and contributes to T cell-mediated cytotoxicity (Nagata and Golstein, 1995). By contrast, the absence of the ~55 receptor for TNF (TNFRl) is associated with the inability to generate an appropriate host defense response against certain patho-

Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13.

Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12-p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children.

Targeting lactate metabolism for cancer therapeutics

Lactate, once considered a waste product of glycolysis, has emerged as a critical regulator of cancer development, maintenance, and metastasis. Indeed, tumor lactate levels correlate with increased metastasis, tumor recurrence, and poor outcome. Lactate mediates cancer cell intrinsic effects on metabolism and has additional non-tumor cell autonomous effects that drive tumorigenesis. Tumor cells can metabolize lactate as an energy source and shuttle lactate to neighboring cancer cells, adjacent stroma, and vascular endothelial cells, which induces metabolic reprogramming. Lactate also plays roles in promoting tumor inflammation and in functioning as a signaling molecule that stimulates tumor angiogenesis. Here we review the mechanisms of lactate production and transport and highlight emerging evidence indicating that targeting lactate metabolism is a promising approach for cancer therapeutics.

Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis.

By searching a database of expressed sequences, we identified a member of the signal transducers and activators of transcription (Stat) family of proteins. Human and murine full-length cDNA clones were obtained and sequenced. The sequence of the human cDNA was identical to the recently published sequence for interleukin-4 (IL-4)-Stat (J. Hou, U. Schindler, W.J. Henzel, T.C. Ho, M. Brasseur, and S. L. McKnight, Science 265:1701-1706, 1994), while the murine Stat6 amino acid and nucleotide sequences were 83 and 84% identical to the human sequences, respectively. Using Stat6-specific antiserum, we demonstrated that Stat6 is rapidly tyrosine phosphorylated following stimulation of appropriate cell lines with IL-4 or IL-3 but is not detectably phosphorylated following stimulation with IL-2, IL-12, or erythropoietin. In contrast, IL-2, IL-3, and erythropoietin induce the tyrosine phosphorylation of Stat5 while IL-12 uniquely induces the tyrosine phosphorylation of Stat4. Inducible tyrosine phosphorylation of Stat6 requires the membrane-distal region of the IL-4 receptor alpha chain. This region of the receptor is not required for cell growth, demonstrating that Stat6 tyrosine phosphorylation does not contribute to mitogenesis.