Illuminating the functional and structural repertoire of human TBC/RABGAPs.

The Tre2-Bub2-Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulators of RABs. The importance of TBC/RABGAPs in the regulation of specific intracellular trafficking routes is now emerging, as is their role in different diseases. Importantly, TBC/RABGAPs act as key regulatory nodes, integrating signalling between RABs and other small GTPases and ensuring the appropriate retrieval, transport and delivery of different intracellular vesicles.

IL-6 Trans-signaling Controls Liver Regeneration After Partial Hepatectomy.

Interleukin-6 (IL-6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL-6 trans-signaling through the soluble IL-6/IL-6R complex is involved in this process. However, the long-term contribution of IL-6 trans-signaling for liver regeneration after PHX is unknown. PHX-induced generation of the soluble IL-6R by ADAM (a disintegrin and metallo) proteases enables IL-6 trans-signaling, in which IL-6 forms an agonistic complex with the soluble IL-6 receptor (sIL-6R) to activate all cells expressing the signal-transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL-6 in complex with membrane-bound IL-6R and gp130 activates classic signaling. Here, we describe the generation of IL-6 trans-signaling mice, which exhibit boosted IL-6 trans-signaling and abrogated classic signaling by genetic conversion of all membrane-bound IL-6R into sIL-6R proteins phenocopying hyperactivation of ADAM-mediated shedding of IL-6R as single substrate. Importantly, although IL-6R deficient mice were strongly affected by PHX, survival and regeneration of IL-6 trans-signaling mice was indistinguishable from control mice, demonstrating that IL-6 trans-signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long-term consequences of global IL-6 signaling inhibition versus IL-6 trans-signaling selective blockade after PHX by IL-6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL-6 blockade and selective inhibition of IL-6 trans-signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL-6 trans-signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL-6 trans-signaling, but not classic signaling, controls liver regeneration following PHX.

The balance of interleukin (IL)-6, IL-6·soluble IL-6 receptor (sIL-6R), and IL-6·sIL-6R·sgp130 complexes allows simultaneous classic and trans-signaling.

Interleukin (IL-)6 is the major pro-inflammatory cytokine within the IL-6 family. IL-6 signals via glycoprotein 130 (gp130) and the membrane-bound or soluble IL-6 receptor (IL-6R), referred to as classic or trans-signaling, respectively. Whereas inflammation triggers IL-6 expression, eventually rising to nanogram/ml serum levels, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) are constitutively present in the upper nanogram/ml range. Calculations based on intermolecular affinities have suggested that systemic IL-6 is immediately trapped in IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes, indicating that sIL-6R and sgp130 constitute a buffer system that increases the serum half-life of IL-6 or restricts systemic IL-6 signaling. However, this scenario has not been experimentally validated. Here, we quantified IL-6·sIL-6R and IL-6·sIL-6R·sgp130 complexes over a wide concentration range. The amounts of IL-6 used in this study reflect concentrations found during active inflammatory events. Our results indicated that most IL-6 is free and not complexed with sIL-6R or sgp130, indicating that the level of endogenous sgp130 in the bloodstream is not sufficient to block IL-6 trans-signaling via sIL-6R. Importantly, addition of the single-domain antibody VHH6, which specifically stabilizes IL-6·sIL-6R complexes but did not bind to IL-6 or sIL-6R alone, drove free IL-6 into IL-6·sIL-6R complexes and boosted trans-signaling but not classic signaling, demonstrating that endogenous sIL-6R has at least the potential to form complexes with IL-6. Our findings indicate that even though high concentrations of sIL-6R and sgp130 are present in human serum, the relative ratio of free IL-6 to IL-6·sIL-6R allows for simultaneous classic and trans-signaling.

The new sulindac derivative IND 12 reverses Ras-induced cell transformation.

The nonsteroidal anti-inflammatory drug Sulindac has chemopreventive and antitumorigenic properties. Its metabolites induce apoptosis and inhibit signaling pathways critical for malignant transformation, including the Ras pathway. Here we show that the new Sulindac derivative IND 12 reverses the phenotype of Ras-transformed MDCK-f3 cells and restores an untransformed epithelioid morphology characterized by growth in monolayers with regular cell-cell adhesions. Moreover, IND 12 treatment induces the expression at membranes of the cell adhesion protein E-cadherin and increases the level of the E-cadherin-bound beta-catenin. As a consequence, IND 12-treated MDCK-f3 cells lose their invasion capacity and regain the ability to aggregate. In the presence of IND 12, MDCK-f3 cells show regenerated expression and activity ratios of the small GTPases Rac and Rho normally found in untransformed MDCK cells. Strikingly, IND 12 treatment decreases the levels of phosphorylated mitogen-activated protein kinases, which are downstream substrates of the Ras-regulated Raf/mitogen-activated protein kinase pathway, and the level of Ras-induced activation of gene expression. Our findings identify a novel drug with high potential in cancer therapy by targeting Ras-induced cell transformation.

The centrosome and mitotic spindle apparatus in cancer and senescence.

Altered cell division is associated with overproliferation and tumorigenesis, however, mitotic aberrations can also trigger antiproliferative responses leading to postmitotic cell cycle exit. Here, we focus on the role of the centrosome and in particular of centrosomal TACC (transforming acidic coiled coil) proteins in tumorigenesis and cellular senescence. We have complied recent evidence that inhibition or depletion of various mitotic proteins which take over key in centrosome and kinetochore integrity and mitotic checkpoint function in sufficient to activate a p53-p21(WAF) driven premature senescence phenotype. These findings have direct implications for proliferative tissue homeostasis as well as for cellular and organismal aging.

Armus is a Rac1 effector that inactivates Rab7 and regulates E-cadherin degradation.

BACKGROUND: Cell-cell adhesion and intracellular trafficking are regulated by signaling pathways from small GTPases of the Rho, Arf, and Rab subfamilies. How signaling from distinct small GTPases are integrated in a given process is poorly understood. RESULTS: We find that a TBC/RabGAP protein, Armus, integrates signaling between Arf6, Rac1, and Rab7 during junction disassembly. Armus binds specifically to activated Rac1 and its C-terminal TBC/RabGAP domain inactivates Rab7. Thus, Armus is a novel Rac1 effector and a bona fide GAP for Rab7 in vitro and in vivo, a unique and previously unreported combination. Arf6 activation efficiently disrupts cell-cell contacts and is known to activate Rac1 and Rab7. Arf6-induced E-cadherin degradation is efficiently blocked by expression of Armus C-terminal domain or after Armus RNAi. Coexpression of Arf6 with dominant-negative Rab7 or Rac1 also inhibits junction disassembly. Importantly, Armus RabGAP expression also prevents EGF-induced scattering in keratinocytes, a process shown here to require Arf6, Rac1, and Rab7 function. To our knowledge, this is the first report to demonstrate a molecular and functional link between Rac1 and Rab7. CONCLUSIONS: Our data indicate that active Rac1 recruits Armus to locally inactivate Rab7 and facilitate E-cadherin degradation in lysosomes. Thus, the integration of Rac1 and Rab7 activities by Armus provides an important regulatory node for E-cadherin turnover and stability of cell-cell contacts.