The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogen and nutrient signals to control cell proliferation and cell size. Hence, mTORC1 is implicated in a large number of human diseases--including diabetes, obesity, heart disease, and cancer--that are characterized by aberrant cell growth and proliferation. Although eukaryotic translation initiation factor 4E-binding proteins (4E-BPs) are critical mediators of mTORC1 function, their precise contribution to mTORC1 signaling and the mechanisms by which they mediate mTORC1 function have remained unclear. We inhibited the mTORC1 pathway in cells lacking 4E-BPs and analyzed the effects on cell size, cell proliferation, and cell cycle progression. Although the 4E-BPs had no effect on cell size, they inhibited cell proliferation by selectively inhibiting the translation of messenger RNAs that encode proliferation-promoting proteins and proteins involved in cell cycle progression. Thus, control of cell size and cell cycle progression appear to be independent in mammalian cells, whereas in lower eukaryotes, 4E-BPs influence both cell growth and proliferation.The mammalian target of rapamycin complex 1 (mTORC1) controls growth (increase in cell mass) and proliferation (increase in cell number) by modulating mRNA translation through phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BP1, 2, and 3) and the ribosomal protein S6 kinases (S6K1 and 2) (1,2). 4E-BPs regulate the translation of a subset of mRNAs by competing with eIF4G for binding to eIF4E, thus preventing the assembly of the eIF4F complex, whereas the S6Ks control the phosphorylation status of a number of translational components (1-3). Rapamycin has been an important tool in understanding mTORC1 signaling; however, it inefficiently and transiently inhibits 4E-BP phosphorylation (4)( fig. S1A). Moreover, we found that rapamycin inhibited proliferation and G1/S cell cycle progression of WT and 4E-BP double knock-out (DKO) mouse embryonic fibroblasts (MEFs) to the same extent, which suggests that its effects are not mediated by 4E-BPs ( fig. S1, B to D). To directly address the role of 4E-BPs in mTORC1 signaling, we depleted raptor, a component of mTORC1 required for substrate binding (5), in these MEFs. 4E-BP DKO MEFs lack all three 4E-BPs as they do not express 4E-BP3 ( fig. S2A) 1A). Wild-type MEFs in which raptor was depleted proliferated more slowly than control cells, whereas raptor-depleted 4E-BP DKO MEFs proliferated at a rate indistinguishable from that of control cells (Fig. 1B). Similarly, in human embryonic kidney (HEK) 293T cells, raptor silencing had a pronounced effect on mTORC1 signaling and proliferation (Figs. 1C and D). The effect of raptor silencing on proliferation, but not mTOR signaling, was attenuated by codepletion of 4E-BPs (Fig. 1D). Thus, mTORC1-dependent proliferation requires 4E-BPs.To further assess the role of 4E-BPs in mTORC1-mediated cell proliferation, we depleted mTOR or rictor (an mTORC2 specific component), i...

The cells and proteases that mediate cigarette smoke-induced emphysema are controversial, with evidence favoring either neutrophils and neutrophil-derived serine proteases or macrophages and macrophage-derived metalloproteases as the important effectors. We recently reported that both macrophage metalloelastase (MMP-12) and neutrophils are required for acute cigarette smoke-induced connective tissue breakdown, the precursor of emphysema. Here we show how these disparate observations can be linked. Both wild-type (MMP-12 +/+) mice and mice lacking MMP-12 (MMP-12 -/-) demonstrated rapid increases in whole-lung nuclear factor-kappaB activation and gene expression of proinflammatory cytokines after cigarette smoke exposure, indicating that a lack of MMP-12 does not produce a global failure to upregulate inflammatory mediators. However, only MMP-12 +/+ mice demonstrated increased whole-lung tumor necrosis factor-alpha (TNF-alpha) protein or release of TNF-alpha from cultured alveolar macrophages exposed to smoke in vitro. Levels of whole-lung E-selectin, an endothelial activation marker, were increased in only MMP-12 +/+ mice. These findings suggest that, acutely, MMP-12 mediates smoke-induced inflammation by releasing TNF-alpha from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil-derived proteases. TNF-alpha release may be a general mechanism whereby metalloproteases drive cigarette smoke-induced inflammation.

Cell-fate decisions remain poorly understood at the chromatin level. Here, we map chromatin remodeling dynamics during induction of pluripotent stem cells. ATAC-seq profiling of MEFs expressing Oct4-Sox2-Klf4 (OSK) reveals dynamic changes in chromatin states shifting from open to closed (OC) and closed to open (CO), with an initial burst of OC and an ending surge of CO. The OC loci are largely composed of genes associated with a somatic fate, while the CO loci are associated with pluripotency. Factors/conditions known to impede reprogramming prevent OSK-driven OC and skew OC-CO dynamics. While the CO loci are enriched for OSK motifs, the OC loci are not, suggesting alternative mechanisms for chromatin closing. Sap30, a Sin3A corepressor complex component, is required for the OC shift and facilitates reduced H3K27ac deposition at OC loci. These results reveal a chromatin accessibility logic during reprogramming that may apply to other cell-fate decisions.