Interleukin-6 mediates PSAT1 expression and serine metabolism in TSC2-deficient cells.

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either TSC1 or TSC2 Cytokine profiling of TSC2-deficient LAM patient-derived cells revealed striking up-regulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed up-regulation of IL-6 transcription and secretion compared to wild-type cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-13C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wild-type cells. Treatment with anti-IL-6 (αIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in Tsc2+/- mice while reducing PSAT1 expression. These data reveal a mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.

HK2 Mediated Glycolytic Metabolism in Mouse Photoreceptors Is Not Required to Cause Late Stage Age-Related Macular Degeneration-Like Pathologies.

Age-related macular degeneration (AMD) is a multifactorial disease of unclear etiology. We previously proposed that metabolic adaptations in photoreceptors (PRs) play a role in disease progression. We mimicked these metabolic adaptations in mouse PRs through deletion of the tuberous sclerosis complex (TSC) protein TSC1. Here, we confirm our previous findings by deletion of the other complex protein, namely TSC2, in rod photoreceptors. Similar to deletion of Tsc1, mice with deletion of Tsc2 in rods develop AMD-like pathologies, including accumulation of apolipoproteins, migration of microglia, geographic atrophy, and neovascular pathologies. Subtle differences between the two mouse models, such as a significant increase in microglia activation with loss of Tsc2, were seen as well. To investigate the role of altered glucose metabolism in disease pathogenesis, we generated mice with simulation deletions of Tsc2 and hexokinase-2 (Hk2) in rods. Although retinal lactate levels returned to normal in mice with Tsc2-Hk2 deletion, AMD-like pathologies still developed. The data suggest that the metabolic adaptations in PRs that cause AMD-like pathologies are independent of HK2-mediated aerobic glycolysis.

Regulation of colonic epithelial cell homeostasis by mTORC1.

Cell signaling important for homeostatic regulation of colonic epithelial cells (CECs) remains poorly understood. Mammalian target of rapamycin complex 1 (mTORC1), a protein complex that contains the serine-threonine kinase mTOR, mediates signaling that underlies the control of cellular functions such as proliferation and autophagy by various external stimuli. We here show that ablation of tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, specifically in intestinal epithelial cells of mice resulted in increased activity of mTORC1 of, as well as increased proliferative activity of, CECs. Such Tsc2 ablation also reduced the population of Lgr5-positive colonic stem cells and the expression of Wnt target genes in CECs. The stimulatory phosphorylation of the kinase Akt and inhibitory phosphorylation of glycogen synthase kinase 3ß were both markedly decreased in the colon of the Tsc2 conditional knockout (CKO) mice. Development of colonic organoids with cryptlike structures was enhanced for Tsc2 CKO mice compared with control mice. Finally, Tsc2 CKO mice manifested increased susceptibility to dextran sulfate sodium-induced colitis. Our results thus suggest that mTORC1 activity promotes the proliferation of, as well as the expression of Wnt target genes in, CECs and thereby contributes to colonic organogenesis and homeostasis.

Haploinsufficiency of Tsc2 Leads to Hyperexcitability of Medial Prefrontal Cortex via Weakening of Tonic GABAB Receptor-mediated Inhibition.

Loss-of-function mutation in one of the tumor suppressor genes TSC1 or TSC2 is associated with several neurological and psychiatric diseases, including autism spectrum disorders (ASDs). As an imbalance between excitatory and inhibitory neurotransmission, E/I ratio is believed to contribute to the development of these disorders, we investigated synaptic transmission during the first postnatal month using the Tsc2+/- mouse model. Electrophysiological recordings were performed in acute brain slices of medial prefrontal cortex. E/I ratio at postnatal day (P) 15-19 is increased in Tsc2+/- mice as compared with wildtype (WT). At P25-30, facilitated GABAergic transmission reduces E/I ratio to the WT level, but weakening of tonic GABAB receptor (GABABR)-mediated inhibition in Tsc2+/- mice leads to hyperexcitability both at single cell and neuronal network level. Short (1 h) preincubation of P25-30 Tsc2+/- slices with baclofen restores the GABABR-mediated inhibition and reduces network excitability. Interestingly, the same treatment at P15-19 leads to weakening of GABABR-mediated inhibition. We hypothesize that a dysfunction of tonic GABABR-mediated inhibition might contribute to the development of ASD symptoms and suggest that GABABR activation within an appropriate time window may be considered as a therapeutic target in ASD.

Therapeutic Targeting of the Secreted Lysophospholipase D Autotaxin Suppresses Tuberous Sclerosis Complex-Associated Tumorigenesis.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by multiorgan hamartomas, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). TSC2 deficiency leads to hyperactivation of mTOR Complex 1 (mTORC1), a master regulator of cell growth and metabolism. Phospholipid metabolism is dysregulated upon TSC2 loss, causing enhanced production of lysophosphatidylcholine (LPC) species by TSC2-deficient tumor cells. LPC is the major substrate of the secreted lysophospholipase D autotaxin (ATX), which generates two bioactive lipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). We report here that ATX expression is upregulated in human renal angiomyolipoma-derived TSC2-deficient cells compared with TSC2 add-back cells. Inhibition of ATX via the clinically developed compound GLPG1690 suppressed TSC2-loss associated oncogenicity in vitro and in vivo and induced apoptosis in TSC2-deficient cells. GLPG1690 suppressed AKT and ERK1/2 signaling and profoundly impacted the transcriptome of these cells while inducing minor gene expression changes in TSC2 add-back cells. RNA-sequencing studies revealed transcriptomic signatures of LPA and S1P, suggesting an LPA/S1P-mediated reprogramming of the TSC lipidome. In addition, supplementation of LPA or S1P rescued proliferation and viability, neutral lipid content, and AKT or ERK1/2 signaling in human TSC2-deficient cells treated with GLPG1690. Importantly, TSC-associated renal angiomyolipomas have higher expression of LPA receptor 1 and S1P receptor 3 compared with normal kidney. These studies increase our understanding of TSC2-deficient cell metabolism, leading to novel potential therapeutic opportunities for TSC and LAM. SIGNIFICANCE: This study identifies activation of the ATX-LPA/S1P pathway as a novel mode of metabolic dysregulation upon TSC2 loss, highlighting critical roles for ATX in TSC2-deficient cell fitness and in TSC tumorigenesis.

Insulin- and Lipopolysaccharide-Mediated Signaling in Adipose Tissue Macrophages Regulates Postprandial Glycemia through Akt-mTOR Activation.

The physiological role of immune cells in the regulation of postprandial glucose metabolism has not been fully elucidated. We have found that adipose tissue macrophages produce interleukin-10 (IL-10) upon feeding, which suppresses hepatic glucose production in cooperation with insulin. Both elevated insulin and gut-microbiome-derived lipopolysaccharide in response to feeding are required for IL-10 production via the Akt/mammalian target of rapamycin (mTOR) pathway. Indeed, myeloid-specific knockout of the insulin receptor or bone marrow transplantation of mutant TLR4 marrow cells results in increased expression of gluconeogenic genes and impaired glucose tolerance. Furthermore, myeloid-specific Akt1 and Akt2 knockout results in similar phenotypes that are rescued by additional knockout of TSC2, an inhibitor of mTOR. In obesity, IL-10 production is impaired due to insulin resistance in macrophages, whereas adenovirus-mediated expression of IL-10 ameliorates postprandial hyperglycemia. Thus, the orchestrated response of the endogenous hormone and gut environment to feeding is a key regulator of postprandial glycemia.

Podocyte-specific deletion of tubular sclerosis complex 2 promotes focal segmental glomerulosclerosis and progressive renal failure.

Obesity can initiate and accelerate the progression of kidney diseases. However, it remains unclear how obesity affects renal dysfunction. Here, we show that a newly generated podocyte-specific tubular sclerosis complex 2 (Tsc2) knockout mouse model (Tsc2Δpodocyte) develops proteinuria and dies due to end-stage renal dysfunction by 10 weeks of age. Tsc2Δpodocyte mice exhibit an increased glomerular size and focal segmental glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis and proteinaceous casts. Podocytes isolated from Tsc2Δpodocyte mice show nuclear factor, erythroid derived 2, like 2-mediated increased oxidative stress response on microarray analysis and their autophagic activity is lowered through the mammalian target of rapamycin (mTOR)-unc-51-like kinase 1 pathway. Rapamycin attenuated podocyte dysfunction and extends survival in Tsc2Δpodocyte mice. Additionally, mTOR complex 1 (mTORC1) activity is increased in podocytes of renal biopsy specimens obtained from obese patients with chronic kidney disease. Our work shows that mTORC1 hyperactivation in podocytes leads to severe renal dysfunction and that inhibition of mTORC1 activity in podocytes could be a key therapeutic target for obesity-related kidney diseases.

Dexmedetomidine Protects Against Oxygen-Glucose Deprivation-Induced Injury Through Inducing Astrocytes Autophagy via TSC2/mTOR Pathway.

Although there is an increment in stroke burden in the world, stroke therapeutic strategies are still extremely limited to a minority of patients. We previously demonstrated that dexmedetomidine (DEX) protects against focal cerebral ischemia via inhibiting neurons autophagy. Nevertheless, the role of DEX in regulating astrocytes autophagic status in oxygen-glucose deprivation, a condition that mimics cerebral ischemia, is still unknown. In this study, we have shown that DEX and DEX + RAPA (autophagy inducer) increased viability and reduced apoptosis of primary astrocytes in oxygen-glucose deprivation (OGD) model compared with DEX + 3-methyladenine (3-MA) (autophagy inhibitor). DEX induced the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1, while reduced the expression of p62 in primary cultured astrocytes through induction of autophagy. In addition, DEX enhanced the expression of tuberous sclerosis complex 2 (TSC2) in primary cultured astrocytes, while reduced the expression of mammalian target of rapamycin (mTOR). In conclusion, our study suggests that DEX exerts a neuroprotection against OGD-induced astrocytes injury via activation of astrocytes autophagy by regulating the TSC2/mTOR signaling pathway, which provides a new insight into the mechanisms of DEX treatment for acute ischemic injury.

Rapamycin-upregulated miR-29b promotes mTORC1-hyperactive cell growth in TSC2-deficient cells by downregulating tumor suppressor retinoic acid receptor ß (RARß).

miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARß) as a target gene of miR-29b. We found that miR-29b directly targeted the 3'UTR of RARß. Forced expression of RARß reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARß expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARß. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARß-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARß/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.

Regulation of Small Intestinal Epithelial Homeostasis by Tsc2-mTORC1 Signaling.

Mammalian target of rapamycin complex 1 (mTORC1), a protein complex containing the serine/threonine kinase mTOR, integrates various growth stimulating signals. mTORC1 is expressed in intestinal epithelial cells (IECs), whereas the physiological roles of this protein complex in homeostasis of IECs remain virtually unknown. We here generated mice, in which tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, was specifically ablated in IECs (Tsc2 CKO mice). Ablation of Tsc2 enhanced the phosphorylation of mTORC1 downstream molecules such as ribosomal S6 protein and 4E-BP1 in IECs. Tsc2 CKO mice manifested the enhanced proliferative activity of IECs in intestinal crypts as well as the promoted migration of these cells along the crypt-villus axis. The mutant mice also manifested the increased apoptotic rate of IECs as well as the increased ectopic Paneth cells, which are one of the major differentiated IECs. In addition, in vitro study showed that ablation of Tsc2 promoted the development of intestinal organoids without epidermal growth factor, while mTORC1 inhibitor, rapamycin, diminished this phenotype. Our results thus suggest that Tsc2-mTORC1 signaling regulates the proliferation, migration, and positioning of IECs, and thereby contributes to the proper regulation of intestinal homeostasis.

Nociceptor Deletion of Tsc2 Enhances Axon Regeneration by Inducing a Conditioning Injury Response in Dorsal Root Ganglia.

Neurons of the PNS are able to regenerate injured axons, a process requiring significant cellular resources to establish and maintain long-distance growth. Genetic activation of mTORC1, a potent regulator of cellular metabolism and protein translation, improves axon regeneration of peripheral neurons by an unresolved mechanism. To gain insight into this process, we activated mTORC1 signaling in mouse nociceptors via genetic deletion of its negative regulator Tsc2. Perinatal deletion of Tsc2 in nociceptors enhanced initial axon growth after sciatic nerve crush, however by 3 d post-injury axon elongation rate became similar to controls. mTORC1 inhibition prior to nerve injury was required to suppress the enhanced axon growth. Gene expression analysis in purified nociceptors revealed that Tsc2-deficient nociceptors had increased activity of regeneration-associated transcription factors (RATFs), including cJun and Atf3, in the absence of injury. Additionally, nociceptor deletion of Tsc2 activated satellite glial cells and macrophages in the dorsal root ganglia (DRG) in a similar manner to nerve injury. Surprisingly, these changes improved axon length but not percentage of initiating axons in dissociated cultures. The pro-regenerative environment in naïve DRG was recapitulated by AAV8-mediated deletion of Tsc2 in adult mice, suggesting that this phenotype does not result from a developmental effect. Consistently, AAV8-mediated Tsc2 deletion did not improve behavioral recovery after a sciatic nerve crush injury despite initially enhanced axon growth. Together, these data show that neuronal mTORC1 activation induces an incomplete pro-regenerative environment in the DRG that improves initial but not later axon growth after nerve injury.

ERK-TSC2 signalling in constitutively-active HRAS mutant HNSCC cells promotes resistance to PI3K inhibition.

OBJECTIVES: The PI3K/AKT/mTOR pathway is frequently altered in head and neck squamous cell cancer (HNSCC), making this pathway a logical therapeutic target. However, PI3K targeting is not universally effective. Biomarkers of response are needed to stratify patients likely to derive benefit and exclude those unlikely to respond. MATERIALS AND METHODS: We examined the sensitivity of cell lines with constitutively-active (G12V mutant) HRAS and wild-type HRAS to PI3K inhibition using flow cytometry and cell viability assays. We then overexpressed and silenced HRAS and measured sensitivity to the PI3K inhibitor BYL719. Immunoblotting was used to determine activation of the PI3K pathway. MEK and mTOR inhibitors were then tested in HRAS mutant cells to determine their efficacy. RESULTS: HRAS mutant cell lines were non-responsive to PI3K inhibition. Overexpression of HRAS led to reduced susceptibility to PI3K inhibition, while knockdown improved sensitivity. Immunoblotting revealed suppressed AKT phosphorylation upon PI3K inhibition in both wild-type and HRAS mutant cell lines, however mutant lines maintained phosphorylation of S6, downstream of mTOR. Targeting mTOR effectively reduced viability of HRAS mutant cells and we subsequently examined the ERK-TSC2-mTOR cascade as a mediator of resistance to PI3K inhibition. CONCLUSIONS: HRAS mutant cells are resistant to PI3K inhibition and our findings suggest the involvement of a signalling intersection of the MAPK and PI3K pathways at the level of ERK-TSC2, leading to persistent mTOR activity. mTOR inhibition alone or in combination with MAPK pathway inhibition may be a promising therapeutic strategy for this subset of HNSCC tumors.

Uncoupling of ER/Mitochondrial Oxidative Stress in mTORC1 Hyperactivation-Associated Skin Hypopigmentation.

Accumulating evidence has described the involvement of mTORC1 signaling in pigmentation regulation; however, the precise mechanism is not fully understood. Here, we generated mice with conditional deletion of the mTORC1 suppressor Tsc2 in melanocytes. It resulted in constitutive hyperactivation of mTORC1 and reduced skin pigmentation. Mechanistically, neither the number of melanocytes nor the expression of melanogenesis-related enzymes was decreased; however, endoplasmic reticulum and mitochondrial oxidative stress and lower melanization in melanosomes were observed. By contrast, abrogation of mTORC1 by rapamycin completely reversed the reduced pigmentation, and alleviation of endoplasmic reticulum stress by SMER28 or 4-phenylbutyrate (PBA) or alleviation of mitochondrial oxidative stress by administration of adenosine triphosphate partially reversed the reduced pigmentation in these mice. In addition, we showed that these mechanisms were involved in reduced pigmentation of TSC2 small interfering RNA-transfected cultured human primary melanocytes and skin lesions of patients with the TSC gene mutation.