A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

Destabilisation of dimeric 14-3-3 proteins as a novel approach to anti-cancer therapeutics.

14-3-3 proteins play a pivotal role in controlling cell proliferation and survival, two commonly dysregulated hallmarks of cancers. 14-3-3 protein expression is enhanced in many human cancers and correlates with more aggressive tumors and poor prognosis, suggesting a role for 14-3-3 proteins in tumorigenesis and/or progression. We showed previously that the dimeric state of 14-3-3 proteins is regulated by the lipid sphingosine, a physiological inducer of apoptosis. As the functions of 14-3-3 proteins are dependent on their dimeric state, this sphingosine-mediated 14-3-3 regulation provides a possible means to target dimeric 14-3-3 for therapeutic effect. However, sphingosine mimics are needed that are not susceptible to sphingolipid metabolism. We show here the identification and optimization of sphingosine mimetics that render dimeric 14-3-3 susceptible to phosphorylation at a site buried in the dimer interface and induce mitochondrial-mediated apoptosis. Two such compounds, RB-011 and RB-012, disrupt 14-3-3 dimers at low micromolar concentrations and induce rapid down-regulation of Raf-MAPK and PI3K-Akt signaling in Jurkat cells. Importantly, both RB-011 and RB-012 induce apoptosis of human A549 lung cancer cells and RB-012, through disruption of MAPK signaling, reduces xenograft growth in mice. Thus, these compounds provide proof-of-principle for this novel 14-3-3-targeting approach for anti-cancer drug discovery.

The GM-CSF receptor family: mechanism of activation and implications for disease.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation.

Sphingosine and FTY720 directly bind pro-survival 14-3-3 proteins to regulate their function.

The dimeric 14-3-3 protein family protects cells from apoptosis by regulating pro-apoptotic molecules. Conversely, the cationic lipid sphingosine is associated with physiological apoptosis and induces apoptosis in its own right by a largely undefined mechanism. We show here that sphingosine and 14-3-3 interact directly in the control of cell death. The binding of sphingosine to 14-3-3 proteins renders them phosphorylatable at the dimer interface, an event that abolishes the pro-survival signalling of 14-3-3. Sphingosine kinase 1 reduces availability of sphingosine for interaction with 14-3-3, thus inhibiting cell death and providing a new mechanistic insight into the role of this enzyme in cell survival and oncogenesis. Importantly, FTY720, a sphingosine analogue with apoptotic activity that is currently in phase III clinical trials for multiple sclerosis, acts in a similar manner to sphingosine in potentiating 14-3-3 phosphorylation. The biological significance of 14-3-3 phosphorylation was demonstrated with a non-phosphorylatable 14-3-3zeta mutant which retarded apoptosis induced by sphingosine and FTY720. These results demonstrate that direct association of sphingosine with 14-3-3 is required for 14-3-3 phosphorylation, and that this axis can control cell fate. Furthermore, these results suggest a new therapeutic activity for FTY720 as an anti-cancer agent based on this mechanism.

The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

Molecular assembly of the ternary granulocyte-macrophage colony-stimulating factor receptor complex.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in certain infectious diseases. Despite the importance of GM-CSF in regulating myeloid cell numbers and function, little is known about the exact composition and mechanism of assembly of the GM-CSF receptor complex. We have now produced soluble forms of the GM-CSF receptor alpha chain (sGMRalpha) and beta chain (sbetac) and utilized GM-CSF, the GM-CSF antagonist E21R (Glu21Arg), and the betac-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor complex. We found that GM-CSF and E21R were able to form low-affinity, binary complexes with sGMRalpha, each having a stoichiometry of 1:1. Importantly, GM-CSF but not E21R formed a ternary complex with sGMRalpha and sbetac, and this complex could be disrupted by E21R. Significantly, size-exclusion chromatography, analytical ultracentrifugation, and radioactive tracer experiments indicated that the ternary complex is composed of one sbetac dimer with a single molecule each of sGMRalpha and of GM-CSF. In addition, a hitherto unrecognized direct interaction between betac and GM-CSF was detected that was absent with E21R and was abolished by BION-1. These results demonstrate a novel mechanism of cytokine receptor assembly likely to apply also to interleukin-3 (IL-3) and IL-5 and have implications for our molecular understanding and potential manipulation of GM-CSF activation of its receptor.

Endogenous interferon-alpha production by differentiating human monocytes regulates expression and function of the IL-2/IL-4 receptor gamma chain.

In vitro monocyte-derived macrophages (MDMac) and synovial fluid macrophages from inflamed joints differ from monocytes in their responses to interleukin 4 (IL-4). While IL-4 can suppress LPS-induced interleukin beta (IL-beta) and tumour necrosis factor alpha (TNF-alpha) production by monocytes, IL-4 can suppress LPS-induced IL-1 beta, but not TNFalpha production by the more differentiated cells. Recently we reported a correlation between the ability of IL-4 to regulate TNFalpha production by monocytes and the expression of the IL-4 receptor gamma chain or gamma common (gamma c chain). Like MDMac, interferon alpha (IFNalpha)-treated monocytes expressed less IL-4 receptor gamma c chain, reduced levels of IL-4-activated STAT6 and IL-4 could not suppress LPS-induced TNFalpha production. In addition, like monocytes and MDMac, IFNalpha-treated monocytes expressed normal levels of the IL-4 receptor alpha chain and IL-4 significantly suppressed LPS-induced IL-1 beta production. With addition of IFNalpha-neutralizing antibodies, the ability of IL-4 to suppress LPS-induced TNFalpha production with prolonged monocyte culture was restored. Detection of IFNalpha in synovial fluids from inflamed joints further implicates IFNalpha in the inability of IL-4 to suppress TNFalpha production by synovial fluid macrophages. This study identifies a mechanism for the differential expression of gamma c and varied responses to IL-4 by human monocytes compared with MDMac.