Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth.

BACKGROUND: Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear. RESULTS: We first investigated the effects of loss of SREBP function in non-transformed cells. Combined ablation of SREBP1 and SREBP2 by siRNA-mediated gene silencing or chemical inhibition of SREBP activation induced endoplasmic reticulum (ER)-stress and engaged the unfolded protein response (UPR) pathway, specifically under lipoprotein-deplete conditions in human retinal pigment epithelial cells. Induction of ER-stress led to inhibition of protein synthesis through increased phosphorylation of eIF2α. This demonstrates for the first time the importance of SREBP in the coordination of lipid and protein biosynthesis, two processes that are essential for cell growth and proliferation. SREBP ablation caused major changes in lipid composition characterized by a loss of mono- and poly-unsaturated lipids and induced accumulation of reactive oxygen species (ROS) and apoptosis. Alterations in lipid composition and increased ROS levels, rather than overall changes to lipid synthesis rate, were required for ER-stress induction.Next, we analyzed the effect of SREBP ablation in a panel of cancer cell lines. Importantly, induction of apoptosis following SREBP depletion was restricted to lipoprotein-deplete conditions. U87 glioblastoma cells were highly susceptible to silencing of either SREBP isoform, and apoptosis induced by SREBP1 depletion in these cells was rescued by antioxidants or by restoring the levels of mono-unsaturated fatty acids. Moreover, silencing of SREBP1 induced ER-stress in U87 cells in lipoprotein-deplete conditions and prevented tumor growth in a xenograft model. CONCLUSIONS: Taken together, these results demonstrate that regulation of lipid composition by SREBP is essential to maintain the balance between protein and lipid biosynthesis downstream of Akt and to prevent resultant ER-stress and cell death. Regulation of lipid metabolism by the Akt/mTORC1 signaling axis is required for the growth and survival of cancer cells

Hypoxia as a key regulator of angiogenesis and inflammation in rheumatoid arthritis: the role of HIF hydroxylases

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a significant impact on patients’ quality of life. One of the well-described features in RA is hypoxia, together with increased infiltration of macrophages into the inflamed joints. Members of the Hypoxia-inducible factor (HIF) family play key roles in activating the transcription of hypoxia regulated genes. Regulation of HIFs, including the enzymes which regulate their stabilisation and transactivation, namely prolyl hydroxylase domain (PHD) enzymes and factor inhibiting HIF-1 (FIH-1), were the main focus of this thesis. This work aimed to shed more light on the influence of monocyte-to-macrophage differentiation on the Hypoxia/HIF axis as well as how PHDs are being regulated during the differentiation process and thus influence the HIF pathway. For this purpose I have used macrophages derived from a monocytic cell line namely THP-1, or from freshly isolated monocytes, derived from peripheral blood mononuclear cells. Monocytes were differentiated to either the classical (M1) or the alternative (M2) macrophages using granulocyte-macrophage colony stimulating factor or macrophage-colony stimulating factor respectively. THP-1 monocytes were differentiated to a M2-like phenotype using phorbol 12-myristate 13-acetate. Possible differences between the two macrophage phenotypes with regard to the Hypoxia/HIF pathway were also investigated in this study. One of the major findings was that macrophage differentiation leads to stabilisation of HIF-1α isoform even in normoxic (20 % O2) conditions. In particular, in PMA-treated THP-1 cells, the normoxic HIF-1α stabilisation was partly due to an increased HIF α gene transcription. Moreover, PHD-2 expression and enzymatic activity were also affected during the differentiation process, and were linked to reduced HIF hydroxylation and increased HIFα protein accumulation. However, expression of downstream HIF dependent angiogenic genes was not affected during the differentiation process, suggesting an additional level of control, mediated possibly by transcriptional inactivation of HIF via asparagine hydroxylation by FIH-1, or inhibition of the HIF α nuclear translocation machinery. Using the M1 and M2 polarised macrophages, there were no significant differences observed between the two phenotypes with regard to the HIF α isoform expression. In both phenotypes downstream gene expression was observed for Bcl2/adenovirus E1B 19d-interactin protein (BNIP3) and ephrinA3 (EPHRINA3), although in the M2 phenotype expression of both genes was significantly greater. Exposure of macrophages to the hypoxia mimetic dimethyloxaloyglycine (DMOG) stabilised both HIF α protein isoforms with no siginificant differences in expression levels, and led to transcription of the HIF α target genes BNIP3 and EphrinA3. Expresion of PHD-2 and PHD-3 were also increased in response to DMOG but more in the M2 macrophages. The effect of HIF stabilisation by DMOG was also studied in two different in vivo mouse models of arthritis; collagen induced arthritis (CIA) and antigen induced arthritis (AIA). HIF activation by DMOG was observed by imaging analysis, using a previously described transgenic mouse oxygen-dependent degradation domain (ODD)-luciferase reporter mice. It was demonstrated that DMOG activates the HIF pathway in vivo, and appears to exert a protective role in the setting of arthritis (reduced paw/knee swelling), possibly by promoting the expansion of the anti-inflammatory M2 macrophage population. Furthermore the effect of DMOG in anaemia of inflammation was also observed in the CIA mouse model. Arthritic mice showed decreased haematocrit levels, which were corrected back to normal levels after DMOG treatment. In conclusion my data contributes to a better understanding of the Hypoxia/HIF pathway in RA, and also suggest an additional possibly protective role of HIF possibly by shifting macrophages towards a less inflammatory phenotype, and also inhibiting anaemia of inflammation. These findings also set the basis for further research that will allow the development of new therapeutic strategies for the treatment of RA.Open Acces