ELMO1 signaling is a promoter of osteoclast function and bone loss.

Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. Here, we identify the cytoplasmic protein ELMO1 as an important 'signaling node' in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the 'ELMO1 interactome' in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis.

Metabolites released from apoptotic cells act as tissue messengers.

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2-4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues.

ShcA regulates thymocyte proliferation through specific transcription factors and a c-Abl-dependent signaling axis.

Signaling via the pre-T-cell receptor (pre-TCR), along with associated signals from Notch and chemokine receptors, regulates the ß-selection checkpoint that operates on CD4(-) CD8(-) doubly negative (DN) thymocytes. Since many hematopoietic malignancies arise at the immature developmental stages of lymphocytes, understanding the signal integration and how specific signaling molecules and distal transcription factors regulate cellular outcomes is of importance. Here, a series of molecular and genetic approaches revealed that the ShcA adapter protein critically influences proliferation and differentiation during ß-selection. We found that ShcA functions downstream of the pre-TCR and p56(Lck) and show that ShcA is important for extracellular signal-regulated kinase (ERK)-dependent upregulation of transcription factors early growth factor 1 (Egr1) and Egr3 in immature thymocytes and, in turn, of the expression and function of the Id3 and E2A helix-loop-helix (HLH) proteins. ShcA also contributes to pre-TCR-mediated induction of c-Myc and additional cell cycle regulators. Moreover, using an unbiased Saccharomyces cerevisiae (yeast) screen, we identified c-Abl as a binding partner of phosphorylated ShcA and demonstrated the relevance of the ShcA-c-Abl interaction in immature thymocytes. Collectively, these data identify multiple modes by which ShcA can fine-tune the development of early thymocytes, including a previously unappreciated ShcA-c-Abl axis that regulates thymocyte proliferation.

Cooperation between Noncanonical Ras Network Mutations.

Cancer develops after the acquisition of a collection of mutations that together create the cancer phenotype. How collections of mutations work together within a cell and whether there is selection for certain combinations of mutations are not well understood. We investigated this problem with a mathematical model of the Ras signaling network, including a computational random mutagenesis. Modeling and subsequent experiments revealed that mutations of the tumor suppressor gene NF1 can amplify the effects of other Ras pathway mutations, including weakly activating, noncanonical Ras mutants. Furthermore, analyzing recently available, large, cancer genomic data sets uncovered increased co-occurrence of NF1 mutations with mutations in other Ras network genes. Overall, these data suggest that combinations of Ras pathway mutations could serve the role of cancer "driver." More generally, this work suggests that mutations that result in network instability may promote cancer in a manner analogous to genomic instability.

Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region.

Pannexin 1 (PANX1) channels mediate release of ATP, a "find-me" signal that recruits macrophages to apoptotic cells; PANX1 activation during apoptosis requires caspase-mediated cleavage of PANX1 at its C terminus, but how the C terminus inhibits basal channel activity is not understood. Here, we provide evidence suggesting that the C terminus interacts with the human PANX1 (hPANX1) pore and that cleavage-mediated channel activation requires disruption of this inhibitory interaction. Basally silent hPANX1 channels localized on the cell membrane could be activated directly by protease-mediated C-terminal cleavage, without additional apoptotic effectors. By serial deletion, we identified a C-terminal region just distal to the caspase cleavage site that is required for inhibition of hPANX1; point mutations within this small region resulted in partial activation of full-length hPANX1. Consistent with the C-terminal tail functioning as a pore blocker, we found that truncated and constitutively active hPANX1 channels could be inhibited, in trans, by the isolated hPANX1 C terminus either in cells or when applied directly as a purified peptide in inside-out patch recordings. Furthermore, using a cysteine cross-linking approach, we showed that relief of inhibition following cleavage requires dissociation of the C terminus from the channel pore. Collectively, these data suggest a mechanism of hPANX1 channel regulation whereby the intact, pore-associated C terminus inhibits the full-length hPANX1 channel and a remarkably well placed caspase cleavage site allows effective removal of key inhibitory C-terminal determinants to activate hPANX1.

Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis.

Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance.

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y(2) as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y(2)-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y(2)(-/-) (also known as P2ry2(-/-)) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y(2)(-/-) mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y(2)-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

Regulation of the Src homology 2 domain-containing inositol 5'-phosphatase (SHIP1) by the cyclic AMP-dependent protein kinase.

Many agents that activate hematopoietic cells use phos pha tidyl ino si tol 3,4,5-trisphosphate (PtdIns 3,4,5-P(3)) to initiate signaling cascades. The SH2 domain-containing inositol 5' phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phos pha tidyl ino si tol 3-kinase and reducing the levels of PtdIns 3,4,5-P(3). Activation of the cyclic AMP-de pend ent protein kinase (PKA) also opposes many of the pro-inflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phos pho ryl a tion with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phos pho ryl a ted by PKA to a stoichiometry of 0.6 mol of PO(4)/mol of SHIP1. In (32)P-labeled HEK-293 cells transfected with SHIP1, stimulation with Sp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Sp-cAMPS) or activation of the beta-adrenergic receptor increased the phos pho ryl a tion state of SHIP1. Inhibition of protein phosphatase activity with okadaic acid also increased the phos pho ryl a tion of SHIP1. Phosphorylation of SHIP1 in vitro or in cells by PKA increased the 5' phosphatase activity of SHIP1 by 2-3-fold. Elevation of Ca(2+) in DT40 cells in response to B cell receptor cross-linking, an indicator of PtdIns 3,4,5-P(3) levels, was markedly blunted by pretreatment with Sp-cAMPS. This effect was absent in SHIP(-/-) DT40 cells showing that the effect of Sp-cAMPS in DT40 cells is SHIP1-de pend ent. Sp-cAMPS also blunted the ability of the B cell receptor to increase the phos pho ryl a tion of Akt in DT40 and A20 cells. Overall, activation of G protein-coupled receptors that raise cyclic AMP cause SHIP1 to be phosphorylated and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 regulation.