Accumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11 and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA).
Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.
Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL promotes the survival of leukemic cells. Heme oxygenase-1 (HO-1) is an inducible stress protein that catalyzes the degradation of heme and has recently been implicated in the regulation of growth and survival of various neoplastic cells. In the present study, we analyzed the expression and role of HO-1 in CML cells. As assessed by Northern and Western blot analysis as well as immunostaining, primary CML cells were found to express HO-1 mRNA and the HO-1 protein in a constitutive manner. Exposure of these cells to the BCR/ABL tyrosine kinase inhibitor STI571 resulted in decreased expression of HO-1 mRNA and protein. In addition, BCR/ABL was found to up-regulate HO-1 promoter activity, mRNA levels, and protein levels in Ba/F3 cells. To investigate the role of HO-1 for survival of primary CML cells, the HO-1 inducer hemin was used. Hemin-induced expression of HO-1 was found to protect CML cells from STI571-induced cell death. In addition, inhibition of HO-1 by zinc-(II)-deuteroporphyrin-IX-2,4-bisethyleneglycol resulted in a substantial decrease of cell viability. Furthermore, overexpression of HO-1 in the CML-derived cell line K562 was found to counteract STI571-induced apoptosis. Together, our data identify HO-1 as a novel BCR/ABL-driven survival molecule and potential target in leukemic cells in patients with CML. The pathogenetic and clinical implications of this observation remain to be elucidated.
By integrating microenvironmental cues, mTOR has evolved as a critical determinant for the maintenance of cellular homeostasis and function. As such, the unfolding biology of mTOR revealed a crucial role in cancer, metabolic diseases and ageing. Now, accumulating evidence suggests that mTOR may also have an important modulatory function in the cellular response to inflammatory cues, such as TNF. Whether or not this also applies to fibroblast-like synoviocytes (FLS), particularly in the context of rheumatoid synovitis, remains elusive.
Objectives
To evaluate the role of mTOR in the rheumatoid synovial tissue response to inflammation (TNF).
Methods
A simplified 3-D model of the synovium was used to evaluate the significance of mTOR activity for the inflammatory mesencyhmal tissue response. FLS viability and proliferation was assessed by Annexin/7-AAD staining and a 3H-Thymidine incooperation assay, respectively. mTOR activity was blocked by Torin-1, which is a well known specific inhibitor of mTOR functions. In addition, RA-FLS (n=5) were exposed to TNF (6 hours) in the presence or absence of Torin-1 and the GeneChip® PrimeView array™ was used for gene expression profiling. Assessment of regulated genes was determined via SAM analysis. The expression of selected candidates was validated by Q-PCR and ELISA. To assess mTOR activity in RA-FLS, immunoblotting (IB) was performed using phosphospecific antibodies to TSC2, mTOR, AKT and S6K.
Results
Stimulation of the 3D synovial organ cultures with TNF resulted in hyperplasia of the lining layer at the surface of the spheres. Strikingly, the mTOR inhibitor Torin-1 prevented TNF-induced FLS proliferation and lining layer hyperplasia. Importantly, Torin-1 alone or in combination with TNF did not affect RA-FLS viability. On the other side, gene expression profiling revealed that inhibition of mTOR results in the enhanced production of NFkB regulated (proinflammatory) genes, such as IL-6, IL8, or MMP1. Finally, immunoblotting of FLS lysates demonstrated that the mTOR pathway is activated by TNF, suggesting that the FLS response to inflammation (e.g. TNF) is controlled by a signalling cascade that involves mTOR.
Conclusions
These studies provide insight into determinants of the synovial tissue response to inflammation and suggest a multifaceted regulatory role for mTOR in arthritis, especially in rheumatoid arthritis.
On The CoverConfocal microscopy image of a ventricular myocyte from an adult rat heart, with nuclei stained with DAPI (blue) and cardiac myosin-binding protein C (green) and telethonin (red) immunolabeled with specific antibodies.Telethonin is localized to the Z-disc regions of cardiac sarcomeres, where its function may be regulated by phosphorylation of two Ser residues in the C-terminal region of the protein.For details see the article by Candasamy et al., pages
