Matricellular Protein SPARCL1 Regulates Blood Vessel Integrity and Antagonizes Inflammatory Bowel Disease.

BACKGROUND: The understanding of vascular plasticity is key to defining the role of blood vessels in physiologic and pathogenic processes. In the present study, the impact of the vascular quiescence marker SPARCL1 on angiogenesis, capillary morphogenesis, and vessel integrity was evaluated. METHODS: Angiogenesis was studied using the metatarsal test, an ex vivo model of sprouting angiogenesis. In addition, acute and chronic dextran sodium sulfate colitis models with SPARCL1 knockout mice were applied. RESULTS: This approach indicated that SPARCL1 inhibits angiogenesis and supports vessel morphogenesis and integrity. Evidence was provided that SPARCL1-mediated stabilization of vessel integrity counteracts vessel permeability and inflammation in acute and chronic dextran sodium sulfate colitis models. Structure-function analyses of purified SPARCL1 identified the acidic domain of the protein necessary for its anti-angiogenic activity. CONCLUSIONS: Our findings inaugurate SPARCL1 as a blood vessel-derived anti-angiogenic molecule required for vessel morphogenesis and integrity. SPARCL1 opens new perspectives as a vascular marker of susceptibility to colitis and as a therapeutic molecule to support blood vessel stability in this disease.

Species-, organ- and cell-type-dependent expression of SPARCL1 in human and mouse tissues.

SPARCL1 is a matricellular protein with anti-adhesive, anti-proliferative and anti-tumorigenic functions and is frequently downregulated in tumors such as colorectal carcinoma or non-small cell lung cancer. Studies have identified SPARCL1 as an angiocrine tumor suppressor secreted by tumor vessel endothelial cells, thereby exerting inhibitory activity on angiogenesis and tumor growth, in colorectal carcinoma. It is unknown whether SPARCL1 may exert these homeostatic functions in all organs and in other species. Therefore, SPARCL1 expression was comparatively analysed between humans and mice in a systematic manner. Murine Sparcl1 (mSparcl1) is most strongly expressed in the lung; expressed at an intermediate level in most organs, including the large intestine; and absent in the liver. In human tissues, SPARCL1 (hSPARCL1) was detected in all organs, with the strongest expression in the stomach, large intestine and lung, mostly consistent with the murine expression pattern. A striking difference between human and murine tissues was the absence of mSparcl1 expression in murine livers, while human livers showed moderate expression. Furthermore, mSparcl1 was predominantly associated with mural cells, whereas hSPARCL1 was detected in both mural and endothelial cells. Human SPARCL1 expression was downregulated in different carcinomas, including lung and colon cancers. In conclusion, this study revealed species-, organ- and cell-type-dependent expression of SPARCL1, suggesting that its function may not be similar between humans and mice.

IFN-γ-response mediator GBP-1 represses human cell proliferation by inhibiting the Hippo signaling transcription factor TEAD.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts important functions in inflammation, infectious diseases, and cancer. The large GTPase human guanylate-binding protein 1 (GBP-1) is among the most strongly IFN-γ-induced cellular proteins. Previously, it has been shown that GBP-1 mediates manifold cellular responses to IFN-γ including the inhibition of proliferation, spreading, migration, and invasion and through this exerts anti-tumorigenic activity. However, the mechanisms of GBP-1 anti-tumorigenic activities remain poorly understood. Here, we elucidated the molecular mechanism of the human GBP-1-mediated suppression of proliferation by demonstrating for the first time a cross-talk between the anti-tumorigenic IFN-γ and Hippo pathways. The α9-helix of GBP-1 was found to be sufficient to inhibit proliferation. Protein-binding and molecular modeling studies revealed that the α9-helix binds to the DNA-binding domain of the Hippo signaling transcription factor TEA domain protein (TEAD) mediated by the 376VDHLFQK382 sequence at the N-terminus of the GBP-1-α9-helix. Mutation of this sequence resulted in abrogation of both TEAD interaction and suppression of proliferation. Further on, the interaction caused inhibition of TEAD transcriptional activity associated with the down-regulation of TEAD-target genes. In agreement with these results, IFN-γ treatment of the cells also impaired TEAD activity, and this effect was abrogated by siRNA-mediated inhibition of GBP-1 expression. Altogether, this demonstrated that the α9-helix is the proliferation inhibitory domain of GBP-1, which acts independent of the GTPase activity through the inhibition of the Hippo transcription factor TEAD in mediating the anti-proliferative cell response to IFN-γ.

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol.

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Downregulation of PERK activity and eIF2α serine 51 phosphorylation by mTOR complex 1 elicits pro-oxidant and pro-death effects in tuberous sclerosis-deficient cells.

Oxidative stress determines cell fate through several mechanisms, among which regulation of mRNA translation by the phosphorylation of the alpha (α) subunit of the translation initiation factor eIF2α at serine 51 (eIF2αP) plays a prominent role. Increased eIF2αP can contribute to tumor progression as well as tumor suppression. While eIF2αP is increased in most cells to promote survival and adaptation to different forms of stress, we demonstrate that eIF2αP is reduced in tuberous sclerosis complex 2 (TSC2)-deficient cells subjected to oxidative insults. Decreased eIF2αP in TSC2-deficient cells depends on reactive oxygen species (ROS) production and is associated with a reduced activity of the endoplasmic reticulum (ER)-resident kinase PERK owing to the hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1). Downregulation of PERK activity and eIF2αP is accompanied by increased ROS production and enhanced susceptibility of TSC2-deficient cells to extrinsic pro-oxidant stress. The decreased levels of eIF2αP delay tumor formation of TSC2-deficient cells in immune deficient mice, an effect that is significantly alleviated in mice subjected to an anti-oxidant diet. Our findings reveal a previously unidentified connection between mTORC1 and eIF2αP in TSC2-deficient cells with potential implications in tumor suppression in response to oxidative insults.

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly.

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2ß phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2ß mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2ß and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.

mTORC2 Balances AKT Activation and eIF2α Serine 51 Phosphorylation to Promote Survival under Stress.

UNLABELLED: The mTOR nucleates two complexes, namely mTOR complex 1 and 2 (mTORC1 and mTORC2), which are implicated in cell growth, survival, metabolism, and cancer. Phosphorylation of the α-subunit of translation initiation factor eIF2 at serine 51 (eIF2αS51P) is a key event of mRNA translation initiation and a master regulator of cell fate during cellular stress. Recent studies have implicated mTOR signaling in the stress response, but its connection to eIF2αS51P has remained unclear. Herein, we report that genetic as well as catalytic inhibition of mTORC2 induces eIF2αS51P. On the other hand, the allosteric inhibitor rapamycin induces eIF2αS51P through pathways that are independent of mTORC1 inactivation. Increased eIF2αS51P by impaired mTORC2 depends on the inactivation of AKT, which primes the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK. The biologic function of eIF2αS51P was characterized in tuberous sclerosis complex (TSC)-mutant cells, which are defective in mTORC2 and AKT activity. TSC-mutant cells exhibit increased PERK activity, which is downregulated by the reconstitution of the cells with an activated form of AKT1. Also, TSC-mutant cells are increasingly susceptible to ER stress, which is reversed by AKT1 reconstitution. The susceptibility of TSC-mutant cells to ER stress is further enhanced by the pharmacologic inhibition of PERK or genetic inactivation of eIF2αS51P. Thus, the PERK/eIF2αS51P arm is an important compensatory prosurvival mechanism, which substitutes for the loss of AKT under ER stress. IMPLICATIONS: A novel mechanistic link between mTOR function and protein synthesis is identified in TSC-null tumor cells under stress and reveals potential for the development of antitumor treatments with stress-inducing chemotherapeutics.

Hepatoprotective, Antioxidant, and Anticancer Effects of the Tragopogon porrifolius Methanolic Extract.

Tragopogon porrifolius (Asteraceae), commonly referred to as white salsify, is an edible herb used in Lebanese folk medicine to treat cancer and liver dysfunction. In this study, we investigated the antioxidant activity of Tragopogon porrifolius methanolic extract, both in vitro and in vivo, in addition to its hepatoprotective and anticancer activities. Total phenolic and flavonoid contents were measured and found to be 37.0 ± 1.40 mg GAE/g and 16.6 ± 0.42 mg QE/g dry weight, respectively. In vitro antioxidant assays revealed an FRAP value of 659 ± 13.8 µmol Fe(2+)/g of extract and DPPH IC50 value 15.2 µg/mL. In rats subjected to CCl4-induced hepatotoxicity, significant increase in CAT, SOD, and GST levels was detected. The highest dose of the extract (250 mg/kg) recorded a fold increase of 1.68 for SOD, 2.49 for GST, and 3.2 for CAT. The extract also showed substantial decrease in AST (57%), ALT (56%), and LDH (65%) levels. Additionally, the extract caused a dose-dependent decrease in cell viability and proliferation. In conclusion, the methanolic extract of T. porrifolius displayed a relatively high antioxidant activity both in vitro and in vivo as well as hepatoprotective potential against liver toxicity in rats and anticancer effect on MDA-MB-231 and Caco-2 cells.