Journal of Molecular Signaling最新文献

Background: AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that is activated when cells experience energy deficiency and conversely suppressed in surfeit of energy supply. AMPK activation improves insulin sensitivity via multiple mechanisms, among which AMPK suppresses mTOR/S6K-mediated negative feedback regulation of insulin signaling.

Results: In the present study we further investigated the mechanism of AMPK-regulated insulin signaling. Our results showed that 5-aminoimidazole-4-carboxamide-1 ribonucleoside (AICAR) greatly enhanced the ability of insulin to stimulate the insulin receptor substrate-1 (IRS1)-associated PI3K activity in differentiated 3T3-F442a adipocytes, leading to increased Akt phosphorylation at S473, whereas insulin-stimulated activation of mTOR was diminished. In 3T3-F442a preadipocytes, these effects were attenuated by expression of a dominant negative mutant of AMPK alpha1 subunit. The enhancing effect of ACIAR on Akt phosphorylation was also observed when the cells were treated with EGF, suggesting that it is regulated at a step beyond IR/IRS1. Indeed, when the cells were chronically treated with AICAR in the absence of insulin, Akt phosphorylation was progressively increased. This event was associated with an increase in levels of phosphatidylinositol -3,4,5-trisphosphate (PIP3) and blocked by Wortmannin. We then expressed the dominant negative mutant of PTEN (C124S) and found that the inhibition of endogenous PTEN per se did not affect phosphorylation of Akt at basal levels or upon treatment with AICAR or insulin. Thus, this result suggests that AMPK activation of Akt is not mediated by regulating phosphatase and tensin homologue (PTEN).

Conclusion: Our present study demonstrates that AMPK exerts dual effects on the PI3K pathway, stimulating PI3K/Akt and inhibiting mTOR/S6K.

The tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors, both angiomyolipomas and renal cell carcinomas. On the other hand, mice-deficient in the DNA repair enzyme OGG1 spontaneously develop adenoma and carcinoma. Downregulation of tuberin results in a marked decrease of OGG1 and accumulation of oxidative DNA damage, (8-oxodG) in cultured cells. In addition, tuberin haploinsufficiency is associated with the loss of OGG1 and accumulation of 8-oxodG in rat kidney tumor. Deficiency in tuberin results in decreased OGG1 and NF-YA protein expression and increased 8-oxodG in kidney tumor from TSC patients. In the current study, molecular mechanisms by which tuberin regulates OGG1 were explored. The deficiency of tuberin was associated with a significant decrease in NF-YA and loss of OGG1 in kidney tumors of Eker rat. Downregulation of tuberin by siRNA resulted in a marked decrease in NF-YA and OGG1 protein expression in human renal epithelial cells. Localization of NF-YA in wild type and tuberin-deficient cells was examined by western blot and immunostaining assays. In wild type cells, NF-YA was detected in the nucleus while in tuberin deficient cells in the cyotoplasm. Introducing adenovirus-expressing tuberin (Ad-TSC2) into tuberin-deficient cells restored the nuclear localization of NF-YA. These data define a novel mechanism of regulation of OGG1 through tuberin. This mechanism may be important in the pathogenesis of kidney tumors in patients with TSC disease.

Background: The human prostacyclin receptor (hIP) undergoes agonist-induced phosphorylation, desensitisation and internalisation and may be recycled to the plasma membrane or targeted for degradation by, as yet, unknown mechanism(s).

Results: Herein it was sought to investigate the turnover of the hIP under basal conditions and in response to cicaprost stimulation. It was established that the hIP is subject to low-level basal degradation but, following agonist stimulation, degradation is substantially enhanced. Inhibition of the lysosomal pathway prevented basal and agonist-induced degradation of the mature species of the hIP (46-66 kDa). Conversely, inhibition of the proteasomal pathway had no effect on levels of the mature hIP but led to time-dependent accumulation of four newly synthesised immature species (38-44 kDa). It was established that both the mature and immature species of the hIP may be polyubiquitinated and this modification may be required for lysosomal sorting of the mature, internalised receptors and for degradation of the immature receptors by the 26S proteasomes through the ER-associated degradation (ERAD) process, respectively. Moreover, these data substantially advance knowledge of the factors regulating processing and maturation of the hIP, a complex receptor subject to multiple post-translational modifications including N-glycosylation, phosphorylation, isoprenylation, palmitoylation, in addition to polyubiquitination, as determined herein.

Conclusion: These findings indicate that the hIP is post-translationally modified by ubiquitination, which targets the immature species to the 26S proteasomal degradation pathway and the mature species to the lysosomal degradation pathway.

Background: Glucocorticoid hormones (GCs) induce apoptosis of leukemic T-cells by transcriptional regulation via the GC receptor, GR. In the human leukemic CEM cell culture model, RCAN1 has been identified as one of the genes that is specifically upregulated only in the GC-sensitive CEM C7-14 cells, but not in the GC-resistant CEM-C1-15 sister cells in correlation with GC-evoked apoptosis. RCAN1 gene encodes two major protein isoforms of the regulator of calcineurin (RCAN1), RCAN1-1 and RCAN1-4 via alternative splicing of exons 1 and 4 respectively, to exons 5-7. Studies reported here evaluated the differential regulation and function of the two transcripts and protein products of RCAN1 by the synthetic GC dexamethasone (Dex), and by modulators of calcium signaling.

Results: Dex selectively upregulates transcript specific for RCAN 1-1 in glucocorticoid (GC)-susceptible human leukemic CEM-C7-14 cells but not in GC-refractory CEM-C1-15 sister cells. Expression of the second major transcript, RCAN1-4, is upregulated by [Ca2+]i inducers, thapsigargin and A23187, but not by Dex, suggesting a mutually exclusive regulatory pathway for both RCAN1 transcripts. GC-mediated upregulation of RCAN1-1 transcript and RCAN1-1 protein was kinase dependent, and was blocked by staurosporine and the p38 MAP kinase inhibitor SB 202190. RCAN1-1 coimmunoprecipitates with calcineurin PP3C and Dex-mediated RCAN1-1 upregulation correlated with reduction in calcineurin PP3C activity.

Conclusion: Data presented here suggest that GCs specifically upregulate RCAN1-1 transcript and protein while inducers of [Ca2+]i selectively upregulate RCAN1-4. GC-mediated increase in RCAN1-1 abundance and binding possibly inhibits calcineurin activity and modulates apoptosis in CEM-C7-14 cells.

Background: Despite intensive effort, currently no effective anti-Ras therapies have successfully reached clinical application. Previous studies suggest that the histone deacetylatse (HDAC) inhibitor romidepsin, which is currently in clinical trials for the treatment of multiple malignancies, can block Ras-dependent signaling and growth transformation. These studies suggest that mutational activation of Ras may be a useful biomarker for sensitivity to romidepsin and that the anti-tumor activity of this HDAC inhibitor may involve inhibition of Ras effector-mediated signaling.

Results: To rigorously assess romidepsin as an antagonist of Ras, we utilized two well-characterized cell models for Ras transformation. We found that romidepsin blocked the anchorage-dependent and -independent growth of NIH 3T3 fibroblasts and RIE-1 epithelial cells transformed by all three Ras isoforms. However, romidepsin treatment also blocked growth transformation caused by other oncoproteins (B-Raf and ErbB2/Neu), suggesting that romidepsin is not selective for Ras. We also observed striking differences in romidepsin-mediated growth inhibition between transformed NIH 3T3 fibroblasts compared to RIE-1 epithelial cells, suggesting that the mechanism by which romidepsin blocks transformation is dependent on cellular context. Finally, we found that romidepsin did not inhibit Ras activation of the ERK and AKT effector pathways in NIH 3T3 and RIE-1 cells, suggesting that romidepsin does not directly antagonize Ras.

Conclusion: Taken together, our results suggest that romidepsin is not selective for Ras-transformed cells and that the anti-tumor activity of romidepsin is not due to direct inhibition of Ras function.

Background: Matrix Metalloproteinase functions in the remodeling of the extracellular matrix that is integral for many normal and pathological processes such as morphogenesis, angiogenesis, tissue repair, and tumor invasion. The tissue inhibitor of the metalloproteinase family including the tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates the activity of multifunctional metalloproteinase. It is known that IL-8, the target gene of NF-kappaB pathway, increases in the melanoma cells. However, it is not clear whether the TIMP-2 expression regulates the NF-kappaB pathway. In this study, we have used stable melanoma cell lines, parental A2058, A2058T2-1 overexpressing TIMP-2, and A2058T2R-7 underexpressing TIMP-2, to determine the TIMP-2 regulation of the NF-kappaB activity.

Results: We found that the IL-8 secretion and IL-8 mRNA expression significantly increased in the A2058T2-1 overexpressing TIMP-2. TIMP-2 overexpressed cells had the lower basal level of IkappaBalpha, the inhibitor of NF-kappaB, compared to the parental A2058 cells. The transcriptional NF-kappaB activity was increased by the TIMP-2 overexpression. In contrast, A2058T2R-7 underexpressing TIMP-2 had the similar NF-kappaB activity as that in the parental A2058 cell. The apoptotic cells induced by TNF were less in TIMP-2 over-expression cells compared to those in the parental A2058 cells. TIMP-2 over-expression was able to protect cells from apoptosis.

Conclusion: Our data demonstrate that the expression level of TIMP-2 protein can directly modulate the NF-kappaB pathway in human melanoma cells.

Background: Most of breast cancers are considered sporadic and modulation of the two major genes BRCA1 and BRCA2 expressions caused by tissue-specific somatic mutations lead to this pathology. The nutritional intake of phytoestrogens seems to reduce the risk of breast cancer and investigation of their potential as anticancer agents has increased. However, the possible mechanisms and signalling pathways of phytoestrogen action in breast cancer prevention remains unknown.

Results: Using Taqman Low Density Array technology, we investigated the BRCA2 loss of function role in sporadic breast cancers and the links existing with soy isoflavones on a panel of nuclear receptor expression. Human breast cell lines (MCF-7, MDA-MB-231, and MCF-10a) were transfected by BRCA2-siRNA and treated with genistein (18.5 muM) or daidzein (78.5 muM) for 72 h. Generating the transitory knockdown of BRCA2 oncosuppressor, we observed different modulations in several nuclear receptor genes such as ER, RAR and RXR, as well as PPARs and VDR according to the studied breast cell line. Additional isoflavone treatments showed different nuclear receptor gene modulation profiles.

Conclusion: Our results seemed to implicate the oncosuppressor BRCA2 and the phytoestrogen pathways in different nuclear gene expressions via an ER-independent manner.

Background: Estradiol (E2) mediates various intracellular signaling cascades from the plasma membrane via several estrogen receptors (ERs). The pituitary is an estrogen-responsive tissue, and we have previously reported that E2 can activate mitogen-activated protein kinases (MAPKs) such as ERK1/2 and JNK1/2/3 in the membrane ERalpha (mERalpha)-enriched GH3/B6/F10 rat pituitary tumor cell line. Phytoestrogens are compounds found in plants and foods such as soybeans, alfalfa sprouts, and red grapes. They are structurally similar to E2 and share a similar mechanism of action through their binding to ERs. Phytoestrogens bind to nuclear ERs with a much lower affinity and therefore are less potent in mediating genomic responses. However, little is known about their ability to act via mERs to mediate nongenomic effects.

Methods: To investigate the activation of different nongenomic pathways, and determine the involvement of mERalpha, we measured prolactin (PRL) release by radio-immunoassay, MAPK activations (ERK1/2 and JNK1/2/3) via a quantitative plate immunoassay, and intracellular [Ca2+] by Fura-2 fluorescence imaging in cells treated with E2 or four different phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol).

Results: Coumesterol and daidzein increased PRL release similar to E2 in GH3/B6/F10 cells, while genistein and trans-resveratrol had no effect. All of these compounds except genistein activated ERK1/2 signaling at 1-10 picomolar concentrations; JNK 1/2/3 was activated by all compounds at a 100 nanomolar concentration. All compounds also caused rapid Ca2+ uptake, though in unique dose-dependent Ca2+ response patterns for several aspects of this response. A subclone of GH3 cells expressing low levels of mERalpha (GH3/B6/D9) did not respond to any phytoestrogen treatments for any of these responses, suggesting that these nongenomic effects were mediated via mERalpha.

Conclusion: Phytoestrogens were much more potent in mediating these nongenomic responses (activation of MAPKs, PRL release, and increased intracellular [Ca2+]) via mERalpha than was previously reported for genomic responses. The unique non-monotonic dose responses and variant signaling patterns caused by E2 and all tested phytoestrogens suggest that complex and multiple signaling pathways or binding partners could be involved. By activating these different nongenomic signaling pathways, phytoestrogens could have significant physiological consequences for pituitary cell functions.

Background: While evidence suggested that the activity states of Protein kinase B (AKT/PKB) and endothelial nitric oxide synthase (eNOS) play an important role in the progression of the Growth Hormone (GH) signal cascade, the implication of the activation of AKT/PKB and eNOS in terms of their function in the signaling pathway was not clear.

Results: Using a specific AKT/PKB inhibitor and a functional proteomic approach, we were able to detect the activities of multiple signal transduction pathway elements, the downstream targets of the AKT/PKB pathway and the modification of those responses by treatment with GH. Inhibiting the AKT/PKB activity reduced or eliminated the activation (phosphorylation) of eNOS. We demonstrated that the progression of the GH signal cascade is influenced by the activity status of AKT and eNOS, wherein the suppression of AKT activity appears to augment the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2) and to antagonize the deactivation (phosphorylation) of cyclin-dependent kinase 2 (CDC2/Cdk1) induced by GH. Phosphorylation of GSK3a/b (glycogen synthase kinase 3), the downstream target of AKT/PKB, was inhibited by the AKT/PKB inhibitor. GH did not increase phosphorylation of ribosomal S6 kinase 1 (RSK1) in normal cells but increases phosphorylation of RSK1 in cells pre-treated with the AKT and eNOS inhibitors.

Conclusion: The MAP kinase and CDC2 kinase-dependent intracellular mechanisms are involved in or are the targets of the GH's action processes, and these activities are probably directly or indirectly modulated by AKT/PKB pathways. We propose that the AKT/PKB-eNOS module likely functions as a negative feedback mediator of GH actions.

Background: Sustained agonist-promoted ubiquitination of beta-arrestin has been correlated with increased stability of the GPCR - beta-arrestin complex. Moreover, abrogation of beta-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results: Herein we report that agonist activation of M1 mAChRs produces a sustained beta-arrestin ubiquitination but no stable co-localization with beta-arrestin. In contrast, sustained ubiquitination of beta-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and beta-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of beta-arrestin following agonist treatment, we sought to determine the effects of beta-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated beta-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of beta-arrestin 2 (YFP-beta-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the beta-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced. Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant.

Conclusion: These findings indicate that ubiquitination of beta-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.