The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB-mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB-responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box-containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB-responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

Leptin, an adipocyte-secreted hormone, is one of the central regulators of body weight homeostasis. In humans and rodents, two major forms of leptin receptors (OB-R) are expressed. The short form (OB-R S ), considered to lack signaling capability, is detected in many organs. In contrast, OB-R long form (OB-R L ) predominates in the hypothalamus, but is also present at low levels in peripheral tissues. Transient transfection experiments have demonstrated that OB-R L transduces an intracellular signaling similar to interleukin (IL)-6 type-cytokine receptors. To define the specificity by which OB-R induces genes and cooperates with signal transduction pathways utilized by other hormones and cytokines, rat and human hepatoma cell lines were generated which stably express human OB-R L . Hepatoma cell lines selected for appreciable levels of OB-R L mRNA display enhanced leptin binding and responded to leptin with an IL-6 receptor-like signaling that includes the activation of STAT proteins, induction of acute-phase plasma proteins, and synergism with IL-1 and tumor necrosis factor-␣. A leptin-mediated recruitment of phosphatidylinositol 3-kinase to insulin receptor substrate-2 was also detected. However, no significant tyrosine phosphorylation of insulin receptor substrate-2 and modulation of the immediate cell response to insulin were observed. The data suggest that OB-R L action in hepatic cells is equivalent to that of IL-6 receptor. However, leptin does not play a specific role in muting insulin action on hepatoma cells and therefore may not contribute to the diabetic symptoms associated with obesity.Leptin, a 16-kDa non-glycosylated secretory protein, is produced by adipose cells, released into the circulation, transported across the blood-brain barrier, and acts in a central manner on the hypothalamus to regulate mammalian energy homeostasis (1-5). Leptin signaling is mediated by the leptin receptor (OB-R), 1 a member of the hematopoietin receptor family that appears most closely related to the signal-transducing subunits of the IL-6 type cytokine receptors (6 -8). In humans and rodents, two predominant forms of OB-R are detected. Both isoforms have identical extracellular domains and ligandbinding affinity, but differ in the intracellular domains which represent alternative splice products. The major OB-R short form (OB-R S ) has a 34-amino acid cytoplasmic domain and is found in many organs. However, despite normal ligand binding activity, OB-R S has been described as being incapable of signaling (9, 10). In contrast, the long form (OB-R L ) containing a 302-amino acid cytoplasmic domain, is primarily expressed in specific nuclei of the hypothalamus (11) and is considered to be the signaling-competent receptor isoform (9, 10, 12).Reverse transcriptase-polymerase chain reaction and RNA protection analyses have revealed that various peripheral organs, including liver, have detectable levels of mRNA encoding the OB-R L . The relative amount ranges from 2 to 11% (liver 5%) of the total OB-R mRNA signal (9). Recent...

The circadian clock regulates biological processes from gene expression to organism behavior in a precise, sustained rhythm that is generated at the unicellular level by coordinated function of interlocked transcriptional feedback loops and post-translational modifications of core clock proteins. CLOCK phosphorylation regulates transcriptional activity, cellular localization and stability; however little is known about the specific residues and enzymes involved. We have identified a conserved cluster of serines that include, Ser431, which is a prerequisite phosphorylation site for the generation of BMAL dependent phospho-primed CLOCK and for the potential GSK-3 phosphorylation at Ser427. Mutational analysis and protein stability assays indicate that this serine cluster functions as a phospho-degron. Through the use of GSK-3 activators/inhibitors and kinase assays, we demonstrate that GSK-3β regulates the degron-site by increasing CLOCK phosphorylation/degradation, which correlates with an increase in the expression of CLOCK responsive promoters. Stabilization of phospho-deficient CLOCK delays the phase of oscillation in synchronized fibroblasts. This investigation begins the characterization of a complex phospho-regulatory site that controls the degradation of CLOCK, a core transcription factor that is essential for circadian behavior.

FET cells, derived from an early-stage colon carcinoma, are nontumorigenic in athymic mice. Stable transfection of a dominant-negative transforming growth factor B (TGFB) type II receptor (DNRII) into FET cells that express autocrine TGFB shows loss of TGFB signaling and increased tumorigenicity in vivo indicating tumor suppressor activity of TGFB signaling in this model. The ability of tumorigenic cells to withstand growth factor and nutrient deprivation stress (GFDS) is widely regarded as a key attribute for tumor formation and progression. We hypothesized that increased tumorigenicity of FET/DNRII cells was due to loss of participation of autocrine TGFB in a ''fail-safe'' mechanism to generate cell death in response to this stress. Here, we document that loss of autocrine TGFB in FET/DNRII cells resulted in greater endogenous cell survival in response to GFDS due to activation of the phosphoinositide 3-kinase (PI3K)/Akt/survivin pathway. Treatment of FET DNRII cells with a PI3K inhibitor (LY294002) inhibited Akt phosphorylation and reduced survivin expression resulting in increased apoptosis in FET/DNRII cells. We also show that exogenous TGFB increased apoptosis in FET cells through repression of the PI3K/Akt/survivin pathway during GFDS. These results indicate that the PI3K/Akt/ survivin pathway is blocked by TGFB signaling and that loss of autocrine TGFB leads to increased cell survival during GFDS through the novel linkage of TGFB-mediated repression of survivin expression. Inhibition of survivin function by dominant-negative approaches showed that this inhibitor of apoptosis family member is critical to cell survival in the FET/DNRII cells, thus indicating the importance of this target for TGFB-mediated apoptosis. [Cancer Res 2008;68(9):3152-60]

The leptin receptor (OB-R) mediates the weight regulatory effects of the adipocyte secreted hormone leptin (OB). Previously we have shown that the long form of OB-R, expressed predominantly in the hypothalamus, can mediate ligand-induced activation of signal transducer and activator of transcription factors 1, 3, and 5 and stimulate transcription via interleukin-6 and hematopoietin receptor responsive gene elements. Here we report that deletion and tyrosine substitution mutagenesis of OB-R identifies two distinct regions of the intracellular domain important for signaling. In addition, granulocyte-colony stimulatory factor receptor/OB-R and OB-R/granulocyte-colony stimulatory factor receptor chimeras are signaling competent and provide evidence that aggregation of two OB-R intracellular domains is sufficient for ligand-induced receptor activation. However, signaling by full-length OB-R appears to be relatively resistant to dominant negative repression by signaling-incompetent OB-R, suggesting that mechanisms exist to permit signaling by the long form of OB-R even in the pretence of excess naturally occurring short form of OB-R.Leptin (OB) is an adipose tissue-derived secreted hormone that is thought to suppress appetite by regulating activities of satiety centers in the brain (1). The weight reducing effects of leptin appear to be mediated by interaction with the leptin receptor (OB-R) 1 in the hypothalamus, a region of the brain implicated in the control of body weight (2-4). In mice, mutations in the genes encoding either OB-R (db) or leptin (ob) result in profound early-onset obesity (5, 6). Multiple splice variants of OB-R mRNAs encoding proteins with different length intracellular domains have been detected (7,8). The mutant allele (db) of the OB-R gene was shown to encode a receptor with a truncated cytoplasmic domain (7,8), and more recent data suggest this receptor is signaling inactive (9). Thus, mounting evidence suggests the ability of leptin to regulate body weight is facilitated by downstream signaling events initiated by ligand-induced OB-R activation.Sequence homology and more recent functional data suggest OB-R is a member of the class I cytokine receptor superfamily (4, 10, 11). Receptors of this class lack intrinsic tyrosine kinase activity and are activated by ligand-induced receptor homo-or hetero-dimerization and in many cases require activation of receptor-associated kinases of the Janus family (JAKs) (12). JAKs associate with the membrane-proximal domain of the intracellular part of the cytokine receptors and serve to initiate signal transduction pathways following ligand-induced receptor activation. Included among the downstream targets of the JAK proteins are members of the STAT (Signal Transducers and Activators of Transcription) family of transcription factors (12). The STATs are DNA binding transcription factors that contain Src homology (SH2) domains that interact with receptor molecules through phosphorylated tyrosine residues. STAT proteins are activated by tyrosine phosphorylation...

Abnormal accumulation and activation of receptor tyrosine kinase Ron (recepteur d'origine nantais) has been demonstrated in a variety of primary human cancers. We show that RNA interference-mediated knockdown of Ron kinase in a highly tumorigenic colon cancer cell line led to reduced proliferation as compared with the control cells. Decreased Ron expression sensitized HCT116 cells to growth factor deprivation stress-induced apoptosis as reflected by increased DNA fragmentation and caspase 3 activation. In addition, cell motility was decreased in Ron knockdown cells as measured by wound healing assays and transwell assays. HCT116 cells are heterozygous for gain of function mutant PIK3CA H1047R. Analysis of signaling proteins that are affected by Ron knockdown revealed that phosphatidylinositol 3-kinase (PI3K) activity of the mutant PI3K as well as AKT phosphorylation was substantially reduced in the Ron knockdown cells compared with the control cells. Moreover, we demonstrated in vivo that knockdown of Ron expression significantly reduced lung metastasis as compared with the control cells in the orthotopic models. In summary, our results demonstrate that Ron plays an essential role in maintaining malignant phenotypes of colon cancer cells through regulating mutant PI3K activity. Therefore, targeting Ron kinase could be a potential strategy for colon cancer treatment, especially in patients bearing gain of function mutant PI3K activity.The receptor tyrosine kinase Ron (recepteur d'origine nantais) belongs to the Met proto-oncogene family (1, 2). Mature Ron is a 180-kDa heterodimer composed of a 40-kDa extracellular ␣-chain and a 150-kDa transmembrane ␤-chain with tyrosine kinase activity (2). Macrophage-stimulating protein is the only ligand that has been identified for Ron (3, 4). Upon ligand binding, Ron dimerizes, becomes autophosphorylated, and transduces a variety of signals that regulate different downstream pathways including Ras/mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), 3 c-Jun N-terminal kinase (JNK), ␤-catenin, and nuclear factor-B (5-12). Ron can be activated through ligand-dependent or -independent mechanisms (3,13,14), which lead to responses important for tumorigenesis and metastasis, including cell scattering, proliferation, motility, and survival (15,16).Ron is normally expressed at relatively low levels in cells of epithelial origin (4). Recent studies have shown that Ron is overexpressed in 47% of breast tumor tissues as compared with benign epithelium and that elevated Ron expression was strongly associated with invasive activity by tumors (17). In addition, Ron is moderately expressed in normal colorectal mucosa, but is significantly increased in the majority of primary human colorectal adenocarcinoma samples (18). Ron overexpression has also been demonstrated in head and neck tumors (19). Furthermore, splice variants of Ron have been identified in human colon cancer. These variants were found to confer constitutive Ron activity, transformation, and tumorige...

The nutrient-sensing mTOR (mammalian Target of Rapamycin) pathway regulates cellular metabolism, growth functions, and proliferation and is involved in age-related diseases including cancer, type 2 diabetes, neurodegeneration and cardiovascular disease. The inhibition of mTOR by rapamycin, or calorie restriction, has been shown to extend lifespan and delays tumorigenesis in several experimental models suggesting that rapamycin may be used for cancer prevention. This requires continuous long-term treatment making oral formulations the preferred choice of administration route. However, rapamycin by itself has very poor water solubility and low absorption rate. Here we describe pharmacokinetic and biological properties of novel nanoformulated micelles of rapamycin, Rapatar. Micelles of Rapatar were rationally designed to increase water solubility of rapamycin to facilitate oral administration and to enhance its absorption. As a result, bioavailability of Rapatar was significantly increased (up to 12%) compared to unformulated rapamycin, which concentration in the blood following oral administration remained below level of detection. We also demonstrated that the new formulation does not induce toxicity during lifetime administration. Most importantly, Rapatar extended the mean lifespan by 30% and delayed tumor development in highly tumor-prone p53−/− mice. Our data demonstrate that water soluble Rapatar micelles represent safe, convenient and efficient form of rapamycin suitable for a long-term treatment and that Rapatar may be considered for tumor prevention.

The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.