Roles And Mechanisms Of MTOR Signaling Pathway In Renal Collecting Duct Potassium Excretion And The Prevention Of Mammary Tumorigenesis By N-3 Polyunsaturated Fatty Acids

Background and aimsHyperkalemia is a common clinical and potentially life-threatening metabolic problem in which serum potassium exceeds 5.5 mmol/l. The most important cause of hyperkalemia is a decrease in renal potassium excretion. Excretion mainly occurs in principal cells of the cortical collecting duct (CCD), a two-step process involving (1) cellular potassium entry across the basolateral membrane of the principal cells via the Na+, K+-ATPase pump and (2) potassium exit across the apical membrane via the renal outer medullary K+ channels (ROMK) that open to allow secretion into an electronegative lumen. The two most important physiological determinants of potassium excretion are the serum aldosterone concentration and the delivery of sodium to the distal nephron. Aldosterone binds to the nuclear mineralocorticoid receptor (MR) within the distal tubule (DT) and the principal cells and activates Na+, K+-ATPase, thereby increasing Na+ reabsorption into the blood and the electronegativity of the lumen, providing a more favorable driving force for the secretion of potassium through ROMK. Aldosterone could also upregulate the ENaC and ROMK in the apical membrane of CCD. Therefore, maintaining homeostasis and function of CCD is critical for potassium secretion. However, the molecular mechanisms through which homeostasis and function of CCD are maintained under physiological conditions and disturbed under pathological conditions are not well understood.Mammalian target of rapamycin (mTOR) is a highly conserved Ser/Thr protein kinase and forms two distinct functional complexes, termed mTOR complex 1 (mTORCl) and mTORC2. mTORC1 integrates diverse signals including nutrients, growth factors, energy and stresses to regulate cell growth, proliferation, survival, and metabolism. In response to these stimuli, mTORCl is activated by two families of ras-related small GTPases, Rheb and Rags. GTP-bound (active) Rheb is suppressed by tuberous sclerosis complex 1/2 (TSC1/2), a functional complex which has GTPase-activating protein (GAP) activity toward Rheb. TSC is an inherited benign tumor syndrome characterized by the formation of multiple hamartomas in a wide array of organs including kidney. Loss of TSC1/2 causes cells and tissues to display constitutive mTORCl activation, contributing to their tumor phenotype.Recent studies have demonstrated that mTOR has emerged as an important modulator of several forms of renal disease including renal regeneration after acute kidney injury, chronic kidney diseases, diabetic nephropathy (DN), polycystic kidney disease (PKD), and renal cell carcinoma. Interestingly, TSC1 was observed to be strongly expressed in CCD, indicating its potentially important roles in CCD function. However, the roles of mTOR in CCD function, renal potassium excretion and hyperkalemia are not known. This study aimed to identify the role of mTORCl in the funtion of collecting ducts and potassium homeostasis.Methods1. Using cre-loxp system, we have successfully created the mice with collecting duct-specific deletion of Tscl, which were termed CDTsc1KO mice.2. Blood and urine biological assessment to assess the effects of mTORCl hyperactivation on the renal function and mineral metabolism.3. Histological and morphological analysis (HE, IHC, IF and TEM) to examine the effects of mTORC1 hyperactivation on the structure and morphological appearance of kidney.4. BrdU incorporation and TUNEL assay to determine the effect of abnormal mTORCl activation on the proliferation and apoptosis of collecting duct cells.5. Collecting duct primary cell culture to comfirm the existence of aldosterone resistance.6、Oral administration of rapamycin to test whether mTORCl-dependent CCD cell injury and dysfunction could be prevented by the mTORCl specific inhibitor rapamycin.Results1、Activation of mTORC1 in collecting ducts causes hyperkalemiaWe generated mice (CDTsc1KO) with a conditionally ablated Tscl gene in the collecting duct (principal cells) using a Cre expression cassette under the control of the Aqp2 (aquaporin 2) promoter. Western blotting showed that TSC1 was only seen in wild-type (WT) littermates, but not in CDTsc1KO mice. mTORCl activity was monitored by assaying the level of phospho-S6. The specific enhancement of phospho-S6 in CCD was observed in CDTsc1KO mice, whereas the levels of phosphorylation of Akt (S473) was stable in CDTsc1KO mice CCD. In summary, CDTsc1KO mice have CD-specific inactivation of the Tscl gene and overactivation of mTORCl signaling.Although we observed no obvious differences between CDTsc1KO mice and WT littermate mice in survival, gross physical appearance or organ morphology before the age of 7 weeks, all CDTsclKO animals died around 8 weeks. Blood and urine biochemical analysis revealed that CDTsclKO mice began to show mild hyperkalemia at the age of 4 weeks. Then, at 8 weeks, severe hyperkalemia and increased serum aldosterone were observed, with significant metabolic acidosis, while plasma creatinine, Na+ and Cl- remained unchanged in CDTsclKO mice as compared with littermate mice.2、mTORCl activation causes columnar cell lesions and detachment in cortical collecting ductsWe first focused on the morphological and histological changes of CCD and kidney after specific deletion of TSC1 in CCD. Although no obvious differences between CDTsclKO mice and WT littermates in terms of kidney weight or global appearance were observed in mice of all ages, CCD began to enlarge at the age of 4 weeks in CDTsclKO mice and the area of some enlarged CCD reached 2.7 mm at the age of 8 weeks. Interestingly, massive columnar cell lesions were observed in CCD of 6-8-week-old CDTsclKO mice. The lesions were characterized by columnar-shaped epithelial cells with prominent apical snouts and secretions (CAPSS) seen at the luminal aspect of the cells. Moreover, detachment of columnar-shaped cells from CCD was also seen in 6-8-week-old CDTsclKO mice. Further study revealed remarkably enhanced expression of N-cadherin and vimentin, and almost abolishment of AQP2 in these cells, suggesting that CCD cell detachment may be due to epithelial-mesenchymal transition-like (EMT-like) phenotypic changes. These observations suggest that mTORCl activation causes columnar cell lesions and cell detachment in CCD.3、Rapamycin restores the phenotypes in CDTsc1KO miceWe next examined whether the phynotype could be prevented by the mTORC1 specific inhibitor rapamycin. Interestingly, after 4 weeks of treatment, the rapamycin-treated CDTsc1KO mice survived for a further 4 weeks and died in one week only when rapamycin administration was stopped. Furthermore, the severe hyperkalemia, increased serum aldosterone and metabolic acidosis were largely diminished after rapamycin treatment. Enhanced expression of N-cadherin and vimentin, enlargement of CCD, massive columnar cell lesions and detachment of CCD cells had almost disappeared. These results indicated that mTORC1-dependent CCD injury and the associated pathological phenotypes could be restored upon inactivation of mTORCl by rapamycin.4、mTORCl activation enhances excessive cell proliferation and apoptosis in CCDsWe next examined cell proliferation in the kidneys of CDTsc1KO mice using BrdU (2 h pulse) labeling. Evidence of enhanced proliferation was observed in 4-week-old mice. Cell apoptosis was also assessed in 8-week-old mice by TdT-mediated dUTP nick end labeling (TUNEL) staining, which showed a significant increase of apoptosis in columnar and detached cells, and sporadic apoptotic cells appeared within the lumen. It is suggested that mTORCl activation resulted in increased cell proliferation and apoptosis in the CCD.5、ER stress is a critical factor for CCD cell lesions and detachment in CDTsc1KO miceWe found that GRP78, which is induced during ER stress, was expressed at higher levels in the CCD of CDTsc1KO mice. Evidence of ER stress was further confirmed by transmission electron microscopy (TEM) analysis. The accumulation of GRP78 in CCD of CDTsc1KO mice decreased after rapamycin treatment, suggesting that elevated ER stress is induced by high mTORCl activity. We next administered 4-phenyl butyric acid (PBA) to reduce ER stress. Oral administration of PBA effectively reduced the accumulation of GRP78 in CCD cells and prevented columnar cell lesions and detachment of CCD cells in 8-week-old CDTsc1KO mice. Accordingly, reduction of ER stress with PBA in CDTsc1KO mice significantly decreased N-cadherin and vimentin expression in CCD cells. These results suggest that high mTORCl-induced ER stress causes CCD columnar cell lesions and detachment in CDTsc1KO mice. However, unlike rapamycin, PBA only slightly reduced the level of plasma potassium, indicating that excessive activation of mTORCl-induced ER stress may contribute to CCD cell lesions and detachment in this model, but does not totally explain the hyperkalemia.6、mTORCl activation depressed ENaC, ROMK expression and Na+, K+-ATPase activity in CCDsWe next investigated the effect of mTORC1 activation on ENaC and ROMK1, two important channels for Na+ reabsorption and K+ secretion in CCD. Using micro dissection, we found that the protein levels of ENaCa and ROMK1 were decreased in CDTsc1KO mice. Activity of Na+, K+-ATPase, which is critical for cellular potassium entry across the basolateral membrane of the principal cells of CCD, was also reduced in CDTsc1KO mice. Importantly, the protein levels of ENaCa and ROMK1 and the activity of Na+, K+-ATPase in CDTsc1KO mice could be restored by rapamycin treatment. These findings indicate that excessive mTORCl activation reduces ENaCa, ROMK1 expression and Na+, K+-ATPase activity in CCD.7、mTORCl negatively regulates SGK1 expression and induces aldosterone resistance in CCDWe finally examined the effect of excessive mTORCl activation SGK1 expression in CCD. We found that the protein and mRNA expression of SGK1 were significantly reduced either in situ or in primary cultured CCD from CDTsc1KO mice. Importantly, rapamycin was able to re-induce the expression of SGK1 mRNA and protein in CCD cells of CDTsc1KO mice. Furthermore, aldosterone deprivation-stimulation elevated the levels of SGKl, ENaCa and ROMK1 in cultured CCD from wild type mice but was unable to increase their levels in cultured CCD from CDTsc1KO mice, suggesting the existence of aldosterone resistance in CDTsclKO mice. Taken together, these results demonstrate that mTORCl negatively regulates SGKl expression and mTORC1 hyperactivation induces aldosterone resistance in CCD.ConclusionOur findings suggest that balanced mTORCl activity is critical for maintaining CD function and potassium homeostasis in the kidney. Loss of TSC1 and activation of mTORC1 in CD causes hyperkalemia. Repressed expression of SGK1 by mTORCl activation subsequently decreased the expression and/or activity of ENaC, ROMK1 and Na+, K+-ATPase in CD, which contributed to the aldosterone resistance and hyperkalemia. CDTsc1KO mice represent a novel model for hyperkalemia induced exclusively by dysfunction of CD.Background and aimsBreast cancer is the most common cancer among women worldwide. Although it has yet to be determined what initially causes the onset of breast cancer, research suggests potential links to dietary habits and fat intake. The contribution to mammary carcinogenesis of dietary omega-3 (n-3) polyunsaturated fatty acids (PUFAs) and omega-6 (n-6) PUFAs has received considerable attention in the literature, with some epidemiological studies suggesting a preventative effect of n-3 PUFAs, and a promoting effect of n-6 PUFAs, on breast cancer. Other studies, however, have failed to demonstrate a statistically significant association between n-3 PUFAs and reduced breast cancer risk. Several confounding factors may account for the inconsistent results, namely:1) wide variations in the amount and source of n-3 PUFAs consumed in each study; 2) the ratio of n-6 to -3 may be more important than the absolute amount of n-3 PUFA, as suggested by animal and human studies; 3) results will depend upon the type and even the bioavailability (triglyceride or ester) of n-3 PUFAs used in the studies; and 4) the length of time of the n-3 PUFA diet will also affect the outcome of any study. Collectively, these inconsistencies preclude definitive conclusions, or in-depth mechanistic investigations, in this area, and to date, a unifying mechanistic hypothesis addressing how n-3 PUFAs suppress breast cancer compared with n-6 PUFAs, is lacking.Mammalian target of rapamycm (mTOR) is a highly conserved Ser/Thr kinase which integrates diverse signals including nutrients, growth factors, energy and stresses to control cell growth, proliferation, survival, and metabolism. mTOR elicits its pleiotropic functions in the context of two functionally distinct signaling complexes, termed mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTORC1, which contains mTOR, mLST8/GpT, Raptor and PRAS40, plays a key role in translation initiation by directly phosphorylating p70 S6 kinase 1 (S6K1) and 4E-BP1, and is sensitive to rapamycin. mTORC2 shares mTOR and mLST8/GpL with mTORCl, but possesses three unique components, namely, rictor, mSinl and PRR5/Protor. Despite the presence of mTOR, mTORC2 is not susceptible to acute rapamycin inhibition. The function of mTORC2 is less clear, but it has been shown to phosphorylate Akt at serine 473 and to regulate cytoskeleton organization and cell motility. As a major cancer drug targets, mTORCl/2 signaling is up-regulated in most of cancers and plays critical roles in carcinogenesis and in the progression of cancers. Recent studies have suggested that n-3 PUFAs can negatively regulate the mTORC1/2 signaling, however, the mechanisms through which n-3 PUFAs regulate mTORC1/2 are unknown.Methods1. We first created MNU-induced rat mammary tumor model, and treated them with a low or high dietary ratio of n-3/n-6 PUFAs respectively, to examine the effect of n-3 PUFAs on the incidence, average tumor weights and multiplicity of mammary tumors.2. Breast tumor cells were treated with various growth factors, and at the same time, with DHA, to assess the effect of DHA on the activity and stability of mTORCl/2, and that on the proliferation, apoptosis and angiogenesis of breast tumor cells.3. We have successfully developed a transgenic mouse model that expresses a gene, fat-1, encoding an n-3 fatty acid desaturase. Since this enzyme can catalyze the conversion of n-6 PUFA to n-3 PUFA by introducing a double bond into fatty acyl chains, transgenic expression of fat-1 enables the host to produce n-3 PUFAs endogenously while concomitantly reducing the levels of n-6 PUFAs. fat-1 transgenic mice will also enable the investigation of the biological properties of n-3 PUFAs without having to incorporate n-3 PUFAs such as docosahexaenoic acid (DHA) in the diet.4. To identify the preventive effects of n-3 PUFAs against breast tumor development as well as to investigate the underlying mechanisms, we developed fat-1 transgenic SCID (severe combined immune deficiency) mice and fat-1 transgenic mouse mammary tumor virus (MMTV, an aggressive breast cancer model) double-hybrid mice. Using these genetic models, combined with a conventional dietary approach, we aimed to investigate the effects of n-3 PUFAs on both mTORCl and mTOR2 pathways and on the mammary tumorigenesis and progression.Results1、Dietary n-3 PUFAs prevent MNU-induced rat mammary tumorigenesis and inhibit mTORCl/2 signaling in vivo.In this study, we found that dietary n-3 PUFAs efficiently reduced the incidence of MNU-induced mammary tumors. Average mammary tumors weights and the number of tumors per mouse were also remarkably reduced in rats treated with high n-3/n-6 PUFAs. Interestingly, high dietary n-3 PUFAs repressed both mTORCl and mTORC2 signaling pathways in viv. We suggest that the decrease of mTORCl/2 signaling by n-3 PUFAs may contribute to their inhibitory effects on mammary carcinogenesis and progression.2、n-3 PUFAs inhibits mTORl/2 signaling in breast cancer cell lines.We first examined whether n-3 PUFA DHA inhibits mTORCl/2 in breast cancer cell lines. DHA rapidly and dose-dependently suppressed insulin-, AA-and amino acids-stimulated mTORCl-directed phosphorylation of S6K1 (T389), S6 (S235/235) and 4E-BP1 and mTORC2-directed phosphorylation of Akt (S473) and its downstream target GSK-3 (S9). In MCF-7 cells transfect with the fat-1 cDNA, the phosphorylation of S6K1 (T389), S6 (S235/235),4EBP1, Akt (S473) and GSK-3 (S9) were significantly reduced. These results suggest that both mTORC1 and mTORC2 signaling pathways are targets of exogenous and endogenous n-3 PUFAs in breast cancer cells.To understand the mechanism through which n-3 PUFA inhibits mTORCl, the roles of AMPK/TSC in this process were examined. Knockdown of AMPK or TSC2 by siRNA did not rescue DHA-induced decrease of phospho-S6 (S235/236). These data indicate that activation of AMPK/TSC2 is not involved in rapid inhibition of mTORCl by DHA. Furthermore, PI3-K/TSC2-independent mechanism may contribute to rapid inhibition of mTORCl/2 by DHA. We finally find that DHA stabilized the mTOR-Raptor interaction and inhibited mTORCl activity. These results indicate that stabilization of mTOR-Raptor complex by DHA may contribute to its inhibitory effects against mTORCl.3、n-3 PUFAs depress mTORCl/2 signaling downstream targets.In MCF-7 and MDA-MB-231 cells, DHA reduced HIF-1a and cyclin D1 expression levels and the secretion of VEGF-a. Moreover, expression of fat-1 decreased VEGF and HIF-la levels in MCF-7 cells. Consistent with these results, inhibition of mTORCl by DHA or fat-1 led to suppression of MCF-7 and MDA-MB-231 cell proliferation and angiogenesi. Similarly, inhibition of mTORC2/Akt (S473) by DHA and fat-1 promoted breast cancer cell apoptosis. Taken together, these results further confirmed that both mTORCl and mTORC2 pathways are targets of n-3 PUFAs and are involved in suppression of breast cancer cell proliferation, survival, angiogenesis and in the potentiation of breast cancer cell apoptosis.4、Endogenously produced n-3 PUFAs inhibits mTORC1/2 signaling and prevents breast tumor growth in xenograft modelsWe established two breast tumor xenograft models producing endogenous n-3 PUFAs:breast cancer MDA-MB-231 cells stably expressing fat-1being subcutaneously implanted into nude mice and fat-1 transgenic SCID (severe combined immune deficiency) mice. Fatty acid composition analysis identified a significantly increased ratio of n-3/n-6 PUFAs in these mice compared with control mice without fat-1. Data show that fat-1 gene expression significantly diminished tumor volume and tumor weight. Importantly, phosphorylation of S6K1 (T389), S6 (S235/236) and Akt (S473) were reduced in tumors with fat-1 gene expression. These results demonstrate that cellular production of n-3 PUFAs and a reduced n-6/n-3 ratio in breast cancer cells inhibit mTORCl/2 signaling and prevent their growth in vivo.5、Reduced mTORC1/2 activities and suppression of mammary tumorigenesis and progression in fat-1 transgenic MMTV-PyVT breast cancer mouse modelWe at last asked whether an increased n-3/n-6 PUFA ratio arising from endogenous production of n-3 PUFAs prevents mammary tumorigenesis and metastasis. fat-1 transgenic MMTV(mouse mammary tumor virus)-PyVT (polyoma virus middle T oncogene) breast cancer mouse were created and fatty acid composition analysis confirmed an increased ratio of n-3/n-6 PUFAs in tumors of PyVT-fat-1 mice compared with PyVT control mice. The number of tumors palpated in PyVT-fat-1 mice was significantly less than the number palpated in PyVT control mice. Moreover, the volume of tumors in PyVT-fat-1 mice was also much less than that in PyVT mice. Furthermore, phosphorylation of S6K1 (T389), S6 (S235/236), 4E-BP1 and Akt (S473), as well as expression of VEGF and HIF-la, were reduced in the tumors of-PyVT-fat-1 mice, and cleaved PARP was significantly enhanced. We also found that repression mTORC2 inthe presence of an increase n-3/n-6 PUFA ratio also prevents breast cancer metastasis, since lung metastasis was observed in PyVT mice while no metastastic tumors were detectable in PyVT-fat-1 mice. These data clearly show that reduction of n-6 PUFAs and enrichment n-3 PUFAs in mouse decreases mTORC1/2 activities and prevents breast carcinogenesis and metastasis.Conclusion1. Both dietary and endogenously prevented breast carcinogenesis, tumor growth and metastasis.2. n-3 PUFAs targeted and inhibited the mTORCl/2 signaling pathways in vitro and in vivo.3. n-3 PUFAs inhibit mTORCl via stabilization of the mTOR-raptor complex.4. Our findings convincingly clarify the causal relationship between n-3 PUFAs and breast cancer prevention and establish mTORCl/2 as a target of n-3 PUFAs.