Regulated in Development and DNA Damage Responses -1 (REDD1) Protein Contributes to Insulin Signaling Pathway in Adipocytes

REDD1 (Regulated in development and DNA damage response 1) is a hypoxia and stress response gene and is a negative regulator of mTORC1. Since mTORC1 is involved in the negative feedback loop of insulin signaling, we have studied the role of REDD1 on insulin signaling pathway and its regulation by insulin. In human and murine adipocytes, insulin transiently stimulates REDD1 expression through a MEK dependent pathway. In HEK-293 cells, expression of a constitutive active form of MEK stabilizes REDD1 and protects REDD1 from proteasomal degradation mediated by CUL4A-DDB1 ubiquitin ligase complex. In 3T3-L1 adipocytes, silencing of REDD1 with siRNA induces an increase of mTORC1 activity as well as an inhibition of insulin signaling pathway and lipogenesis. Rapamycin, a mTORC1 inhibitor, restores the insulin signaling after downregulation of REDD1 expression. This observation suggests that REDD1 positively regulates insulin signaling through the inhibition of mTORC1 activity. In conclusion, our results demonstrate that insulin increases REDD1 expression, and that REDD1 participates in the biological response to insulin.     

Roles for peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivators 1α and 1β in regulating response of white and brown adipocytes to hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.     

Interplay between pVHL and mTORC1 pathways in clear-cell renal cell carcinoma

mTOR complex 1 (mTORC1) is implicated in cell growth control and is extensively regulated. We previously reported that in response to hypoxia, mTORC1 is inhibited by the protein regulated in development and DNA damage response 1 (REDD1). REDD1 is upregulated by hypoxia-inducible factor (HIF)-1, and forced REDD1 expression is sufficient to inhibit mTORC1. REDD1-induced mTORC1 inhibition is dependent on a protein complex formed by the tuberous sclerosis complex (TSC)1 and 2 (TSC2) proteins. In clear-cell renal cell carcinoma (ccRCC), the von Hippel-Lindau (VHL) gene is frequently inactivated leading to constitutive activation of HIF-2 and/or HIF-1, which may be expected to upregulate REDD1 and inhibit mTORC1. However, mTORC1 is frequently activated in ccRCC, and mTORC1 inhibitors are effective against this tumor type; a paradox herein examined. REDD1 was upregulated in VHL-deficient ccRCC by in silico microarray analyses, as well as by quantitative real-time PCR, Western blot, and immunohistochemistry. Vhl disruption in a mouse model was sufficient to induce Redd1. Using ccRCC-derived cell lines, we show that REDD1 upregulation in tumors is VHL dependent and that both HIF-1 and HIF-2 are, in a cell-type-dependent manner, recruited to, and essential for, REDD1 induction. Interestingly, whereas mTORC1 is responsive to REDD1 in some tumors, strategies have evolved in others, such as mutations disrupting TSC1, to subvert mTORC1 inhibition by REDD1. Sequencing analyses of 77 ccRCCs for mutations in TSC1, TSC2, and REDD1, using PTEN as a reference, implicate the TSC1 gene, and possibly REDD1, as tumor suppressors in sporadic ccRCC. Understanding how ccRCCs become refractory to REDD1-induced mTORC1 inhibition should shed light into the development of ccRCC and may aid in patient selection for molecular-targeted therapies.     

HIF-1beta determines ABCA1 expression under hypoxia in human macrophages

ATP-binding cassette transporter A1 plays (ABCA1) a major role in reverse cholesterol transport, a process closely related to atherogenesis. In the thickening atherosclerotic lesions lipid loaded macrophages are exposed to regions of local hypoxia that may influence reverse cholesterol transport. Here we studied the effect of hypoxia on ABCA1 regulation and cholesterol efflux in human macrophages.We found that the hypoxia-inducible factor 1 (HIF-1) specifically binds to the HIF-1 response element of the ABCA1 promoter and the HIF-1 complex increases ABCA1 promoter activity along with ABCA1 expression. Primary human macrophages exposed to hypoxia or expressing constitutively active HIF-1alpha responded with a potent change in ABCA1 expression, which showed a strong correlation with HIF-1beta expression (r: 0.95–0.91). Moreover, ABCA1-mediated cholesterol efflux was also found to be regulated by HIF-1beta under hypoxia. In vivo, in macrophages prepared from human atherosclerotic lesions ABCA1 levels showed a strong correlation with HIF-1beta expression. This in vivo regulatory mechanism was confirmed in human pre-eclamptic placentas, a clinical condition with severe local hypoxia.These results demonstrate that HIF-1beta availability determines ABCA1 expression and cholesterol efflux in macrophages under hypoxia and may contribute to the interpersonal variability of atherosclerotic lesion progression.     

Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes

Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1alpha (HIF-1alpha), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H2O2)-induced dysregulation of adiponectin and PAI-1 production. H2O2 treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer binding protein (C/EBPalpha), but had no effect on HIF-1alpha, whereas hypoxia stabilized HIF-1alpha and decreased expression of C/EBPalpha, but not PPARgamma. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases.     

Hypoxic condition- and high cell density-induced expression of Redd1 is regulated by activation of hypoxia-inducible factor-1alpha and Sp1 through the phosphatidylinositol 3-kinase/Akt signaling pathway

Redd1, a recently discovered stress-response gene, is regulated by hypoxia via hypoxia-inducible factor 1 (HIF-1) and by DNA damage via p53/p63; however, the signaling pathway by which its expression is induced by hypoxia has not been elucidated. In the present study, we demonstrated that the expression of Redd1 in response to hypoxia (1% O(2)), hypoxia-mimetic agent, cobalt chloride (CoCl(2)) and high cell density (HCD) requires coactivation of HIF-1alpha and Sp1. CoCl(2) and HCD induced the activation of HIF-1alpha and Sp1 in HeLa cells, and siRNAs targeting HIF-1alpha and Sp1 abrogated Redd1 expression. Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 and by a dominant-negative PI3K mutant reduced the expression of Redd1 and activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Also, suppression of Akt activation blocked the expression of Redd1 and the activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Furthermore, we found that the induction of Redd1 expression by CoCl(2) can be mediated by activation of Sp1 in HIF-1alpha-deficient cells but that a higher level of Redd1 expression is achieved when these cells are transfected with HIF-1alpha. These results demonstrate that hypoxic condition-and HCD-induced expression of Redd1 is mediated by coactivation of Sp1 and HIF-1alpha downstream of the PI3K/Akt signaling pathway.     

REDD1, an inhibitor of mTOR signalling,is regulated by the CUL4A–DDB1 ubiquitin ligase

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia‐inducible factor‐1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post‐translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin‐mediated degradation mediated by the CUL4A–DDB1–ROC1–β‐TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3β. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.