Silencing RIPK1/mTORC1 signalling attenuated the inflammation and oxidative stress in diabetic cardiomyopathy.

BACKGROUND: Diabetes cardiomyopathy (DCM) is one of the major risk factors for the heart failure of the diabetic patients. RIPK1 maybe participate in the regulation of the oxidative stress and inflammation during DCM. METHODS: H&E and Masson staining were utilized to assess the inflammation and fibrosis in myocardial tissues. CCK-8 and TUNEL staining were utilized to analyze cell viability and apoptosis, respectively. SOD activity and MDA content were detected utilizing the kits. The formation of autophagosomes was measured by immunofluorescence assay. RESULTS: RIPK1 and RPTOR (a component of mTORC1) expression and oxidative stress level were upregulated, but autophagy was decreased in the myocardial tissues of DCM rat characterized by the high body weight and blood glucose, abnormal cardiac function, myocardial inflammation and fibrosis. High glucose (HG) treatment resulted in cell viability and autophagy level decreasing, inflammatory cytokines expression increasing and oxidative stress increasing in cardiac fibroblasts (CFs). Meanwhile, RIPK1 and RPTOR expression also was increased in HG-treated cells. HG-induced CFs apoptosis, inflammation, oxidative stress and the inhibition of HG to cell viability and autophagy was partly reversed by the inhibitor of RIPK1 and mTORC1. CONCLUSION: Overall, RIPK1/mTORC1 signalling suppression improved HG-induced apoptosis, inflammation and oxidative stress through activation autophagy. These data provided a reliable evidence that RIPK1 may be a potential target for DCM therapeutic.

Activation of mTORC1 Signaling Cascade in Hippocampus and Medial Prefrontal Cortex Is Required for Antidepressant Actions of Vortioxetine in Mice.

BACKGROUND: Although thought of as a multimodal-acting antidepressant targeting the serotonin system, more molecules are being shown to participate in the antidepressant mechanism of vortioxetine. A previous report has shown that vortioxetine administration enhanced the expression of rapamycin complex 1 (mTORC1) in neurons. It has been well demonstrated that mTORC1 participates in not only the pathogenesis of depression but also the pharmacological mechanisms of many antidepressants. Therefore, we speculate that the antidepressant mechanism of vortioxetine may require mTORC1. METHODS: Two mouse models of depression (chronic social defeat stress and chronic unpredictable mild stress) and western blotting were first used together to examine whether vortioxetine administration produced reversal effects against the chronic stress-induced downregulation in the whole mTORC1 signaling cascade in both the hippocampus and medial prefrontal cortex (mPFC). Then, LY294002, U0126, and rapamycin were used together to explore whether the antidepressant effects of vortioxetine in mouse models of depression were attenuated by pharmacological blockade of the mTORC1 system. Furthermore, lentiviral-mTORC1-short hairpin RNA-enhanced green fluorescence protein (LV-mTORC1-shRNA-EGFP) was adopted to examine if genetic blockade of mTORC1 also abolished the antidepressant actions of vortioxetine in mice. RESULTS: Vortioxetine administration produced significant reversal effects against the chronic stress-induced downregulation in the whole mTORC1 signaling cascade in both the hippocampus and mPFC. Both pharmacological and genetic blockade of the mTORC1 system notably attenuated the antidepressant effects of vortioxetine in mice. CONCLUSIONS: Activation of the mTORC1 system in the hippocampus and mPFC is required for the antidepressant actions of vortioxetine in mice.

Glucose metabolic upregulation via phosphorylation of S6 ribosomal protein affects tumor progression in distal cholangiocarcinoma.

BACKGROUND: The prognosis of distal cholangiocarcinoma (dCCA) remains poor; thus, the identification of new therapeutic targets is warranted. Phosphorylated S6 ribosomal protein indicates a mammalian target of rapamycin complex 1 (mTORC1) activity, and mTORC1 plays a central role in controlling cell growth and regulating glucose metabolism. We aimed to clarify the effect of S6 phosphorylation on tumor progression and the glucose metabolic pathway in dCCA. METHODS: Thirty-nine patients with dCCA who underwent curative resection were enrolled in this study. S6 phosphorylation and the expression of GLUT1 were evaluated by immunohistochemistry, and their relationship with clinical factors was investigated. The effect of S6 phosphorylation on glucose metabolism with PF-04691502 treatment, an inhibitor of S6 phosphorylation, was examined in cancer cell lines by Western blotting and metabolomics analysis. Cell proliferation assays were performed with PF-04691502. RESULTS: S6 phosphorylation and the expression of GLUT1 were significantly higher in patients with an advanced pathological stage. Significant correlations between GLUT1 expression, S6 phosphorylation, and SUV-max of FDG-PET were shown. In addition, cell lines with high S6 phosphorylation levels showed high GLUT1 levels, and the inhibition of S6 phosphorylation reduced the expression of GLUT1 on Western blotting. Metabolic analysis revealed that inhibition of S6 phosphorylation suppressed pathways of glycolysis and the TCA cycle in cell lines, and then, cell proliferation was effectively reduced by PF-04691502. CONCLUSION: Upregulation of glucose metabolism via phosphorylation of S6 ribosomal protein appeared to play a role in tumor progression in dCCA. mTORC1 may be a therapeutic target for dCCA.

A new glance at autophagolysosomal-dependent or -independent function of transcriptional factor EB in human cancer.

Autophagy-lysosome system plays a variety of roles in human cancers. In addition to being implicated in metabolism, it is also involved in tumor immunity, remodeling the tumor microenvironment, vascular proliferation, and promoting tumor progression and metastasis. Transcriptional factor EB (TFEB) is a major regulator of the autophagy-lysosomal system. With the in-depth studies on TFEB, researchers have found that it promotes various cancer phenotypes by regulating the autophagolysosomal system, and even in an autophagy-independent way. In this review, we summarize the recent findings about TFEB in various types of cancer (melanoma, pancreatic ductal adenocarcinoma, renal cell carcinoma, colorectal cancer, breast cancer, prostate cancer, ovarian cancer and lung cancer), and shed some light on the mechanisms by which it may serve as a potential target for cancer treatment.

Age-Related Decline in Nrf2/ARE Signaling Is Associated with the Mitochondrial DNA Damage and Cognitive Impairments.

In this research, we compared the cognitive parameters of 2-, 7-, and 15-month-old mice, changes in mitochondrial DNA (mtDNA) integrity and expression of genes involved in the nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway. We showed an age-related decrease in the Nfe2l2 expression in the cerebral cortex, not in the hippocampus. At the same time, we find an increase in the mtDNA copy number in the cerebral cortex, despite the lack of an increase in gene expression, which is involved in the mitochondrial biogenesis regulation. We suppose that increase in mtDNA content is associated with mitophagy downregulation. We supposed that mitophagy downregulation may be associated with an age-related increase in the mtDNA damage. In the hippocampus, we found a decrease in the Bdnf expression, which is involved in the pathways, which play an essential role in regulating long-term memory formation. We showed a deficit of working and reference memory in 15-month-old-mice in the water Morris maze, and a decrease in the exploratory behavior in the open field test. Cognitive impairments in 15-month-old mice correlated with a decrease in Bdnf expression in the hippocampus, Nfe2l2 expression, and an increase in the number of mtDNA damage in the cerebral cortex. Thus, these signaling pathways may be perspective targets for pharmacological intervention to maintain mitochondrial quality control, neuronal plasticity, and prevent the development of age-related cognitive impairment.

The gene for the lysosomal protein LAMP3 is a direct target of the transcription factor ATF4.

Autophagy and lysosomal activities play a key role in the cell by initiating and carrying out the degradation of misfolded proteins. Transcription factor EB (TFEB) functions as a master controller of lysosomal biogenesis and function during lysosomal stress, controlling most but, importantly, not all lysosomal genes. Here, we sought to better understand the regulation of lysosomal genes whose expression does not appear to be controlled by TFEB. Sixteen of these genes were screened for transactivation in response to diverse cellular insults. mRNA levels for lysosomal-associated membrane protein 3 (LAMP3), a gene that is highly up-regulated in many forms of cancer, including breast and cervical cancers, were significantly increased during the integrated stress response, which occurs in eukaryotic cells in response to accumulation of unfolded and misfolded proteins. Of note, results from siRNA-mediated knockdown of activating transcription factor 4 (ATF4) and overexpression of exogenous ATF4 cDNA indicated that ATF4 up-regulates LAMP3 mRNA levels. Finally, ChIP assays verified an ATF4-binding site in the LAMP3 gene promoter, and a dual-luciferase assay confirmed that this ATF4-binding site is indeed required for transcriptional up-regulation of LAMP3 These results reveal that ATF4 directly regulates LAMP3, representing the first identification of a gene for a lysosomal component whose expression is directly controlled by ATF4. This finding may provide a key link between stresses such as accumulation of unfolded proteins and modulation of autophagy, which removes them.

Methionine controls insulin/mammalian target of rapamycin complex 1 activity by modulating tuberous sclerosis complex 2 stability.

Tuberous sclerosis complex 2 (TSC2) is a tumor-suppressor protein that is partially regulated by insulin, energy, oxygen, and growth factors. Mutations in the TSC2 gene and loss of TSC2 promote cell growth by the mammalian target of rapamycin complex 1 (mTORC1) activation. Furthermore, S-adenosylmethionine (SAM) sensor upstream of mTORC1 indirectly inhibits mTORC1 activity via the methionine metabolite SAM. Here, we investigated the effects of methionine on insulin/TSC2/mTORC1 activity. Our results showed that methionine affected TSC2 stability and abolished TSC2 localization to the lysosome. Moreover, activation of insulin signaling contributed to TSC2 degradation in a methionine deprivation-dependent manner. Thus, methionine and insulin crosstalk occurred via TSC2.

PPP1CA/YAP/GS/Gln/mTORC1 pathway activates retinal Müller cells during diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-loss complication caused by diabetes with high prevalence. During DR, the retinal microvascular injury and neurodegeneration derived from chronic hyperglycemia have attracted global attention to retinal Müller cells (RMCs), the major macroglia in the retina contributes to neuroprotection. Protein Phosphatase 1 Catalytic Subunit Alpha (PPP1CA) dephosphorylates the transcriptional coactivator Yes-associated protein (YAP) to promote the transcription of glutamine synthetase (GS). GS catalyzes the transformation of neurotoxic glutamate (Glu) into nontoxic glutamine (Gln) to activate the mammalian target of rapamycin complex 1 (mTORC1), which promotes the activation of RMCs. In this study, in vitro MIO-M1 cell and in vivo mouse high-fat diet and streptozotocin (STZ)-induced diabetic model to explore the role of the PPP1CA/YAP/GS/Gln/mTORC1 pathway on the activation of MRCs during DR. Results showed that PPP1CA promoted the dephosphorylation and nuclear translocation of YAP in high glucose (HG)-exposed MIO-M1 cells. YAP transcribed GS in HG-exposed MIO-M1 cells in a TEAD1-dependent and PPP1CA-dependent way. GS promoted the biosynthesis of Gln in HG-exposed MIO-M1 cells. Gln activated mTORC1 instead of mTORC2 in HG-exposed MIO-M1 cells. The proliferation and activation of HG-exposed MIO-M1 cells were PPP1CA/YAP/GS/Gln/mTORC1-dependent. Finally, RMC proliferation and activation during DR were inhibited by the PPP1CA/YAP/GS/Gln/mTORC1 blockade. The findings supplied a potential idea to protect RMCs and alleviate the development of DR.

miR-196b-5p controls adipocyte differentiation and lipogenesis through regulating mTORC1 and TGF-ß signaling.

MicroRNAs have been reported to play a role in adipogenesis and obesity. This study was performed to investigate the role of miR-196b-5p in adipogenesis and the mechanism involved. The data revealed that miR-196b-5p expression increased in primary or established marrow stromal progenitor cells after adipogenic treatment. Supplementing miR-196b-5p in the progenitor cells stimulated adipogenic differentiation and lipogenesis, along with the induction of adipogenic and lipogenic factors. Conversely, inhibition of endogenous miR-196b-5p blocked adipogenesis and lipogenesis. Tuberous sclerosis 1 (Tsc1) and transforming growth factor-ß receptor 1 (TGFBR1) were demonstrated to be the direct target genes of miR-196b-5p. Supplementing miR-196b-5p activity in progenitor cells reduced the protein level of TSC1 and activated mammalian target of rapamycin complex 1 (mTORC1) signaling. We further demonstrated that the perturbation of TSC1 in progenitor cells altered the trend of adipogenic differentiation and lipogenesis. Overexpression of Tsc1 or inactivation of mTORC1 signaling attenuated the stimulation of adipogenic differentiation and lipogenesis by miR-196b-5p. Overexpression of Tgfbr1 also partially blocked the adipogenic effect of miR-196b-5p. Further investigations demonstrated that zinc finger E-box-binding homeobox 1 (ZEB1) transcriptionally upregulated miR-196b-5p expression. The current study suggests that miR-196b-5p promotes adipogenic differentiation and lipogenesis in progenitor cells through targeting TSC1 and TGFBR1 and therefore regulating mTORC1 and TGF-ß signaling.

Maslinic acid activates mTORC1 and human TGR5 and induces skeletal muscle hypertrophy.

Maslinic acid, a naturally occurring pentacyclic triterpene in more than 30 plants (including olives), reportedly increases human muscle mass and muscle strength; however, the underlying molecular mechanism remains unknown. C57BL/6J mice were fed a standard diet or supplemented with 0.27% maslinic acid for 4 weeks, and their skeletal muscle mass was measured. Mice that consumed maslinic acid displayed significant increases in gastrocnemius and soleus muscle mass. Cultured mouse-C2C12 skeletal muscle cells were treated with mammalian target of rapamycin complex 1 (mTORC1) or protein kinase b (Akt) inhibitor, and protein synthesis was quantified. Maslinic acid accelerated protein synthesis via mTORC1 activation independent of Akt. Furthermore, maslinic acid activated human Takeda G protein-coupled receptor 5 (TGR5) more strongly than mouse TGR5, augmenting the expression of several genes related to muscular hypertrophy. Maslinic acid activated mTORC1 and human TGR5, implying its contribution to human muscular hypertrophy through these effects.

Amino Acids in Autophagy: Regulation and Function.

Autophagy is a dynamic process in which the eukaryotic cells break down intracellular components by lysosomal degradation. Under the normal condition, the basal level of autophagy removes damaged organelles, misfolded proteins, or protein aggregates to keep cells in a homeostatic condition. Deprivation of nutrients (e.g., removal of amino acids) stimulates autophagy activity, promoting lysosomal degradation and the recycling of cellular components for cell survival. Importantly, insulin and amino acids are two main inhibitors of autophagy. They both activate the mTOR complex 1 (mTORC1) signaling pathway to inhibit the autophagy upstream of the uncoordinated-51 like kinase 1/2 (ULK1/2) complex that triggers autophagosome formation. In particular, insulin activates mTORC1 via the PI3K class I-AKT pathway; while amino acids activate mTORC1 either through the PI3K class III (hVps34) pathway or through a variety of amino acid sensors located in the cytosol or lysosomal membrane. These amino acid sensors control the translocation of mTORC1 from the cytosol to the lysosomal surface where mTORC1 is activated by Rheb GTPase, therefore regulating autophagy and the lysosomal protein degradation.

Aging and Protein Kinases.

Recently, aging has been tried to be explained with large numbers of theories, but none of them can elucidate the changes occurring in the aging process alone. A unified theory encompassing the mechanisms of genetic factors and repair systems in aging is becoming increasingly required. Almost 37 protein kinases contribute to all processes of aging and senescence. Furthermore, these kinases not only regulate the large number of metabolic pathways related to aging processes, but also control these pathways through 12 checkpoints. Thus, in this chapter, the metabolic targets of protein kinases signal transduction pathways were discussed in terms of the aging perspective under five headings, which are the indispensable stages of the aging process. Although the most popular classical aging theories have been stated as DNA damage theory, mitochondrial theory, free radical theory, and telomere theory, it was concluded that the aging process is controlled by protein kinases regardless of the different theories.

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.

The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy.

Excessive compensatory nephron hypertrophy (CNH) has been implicated in setting the stage for progressive nephron damage. Lack of a class III phosphatidylinositol 3-kinase (Pik3c3) inhibitor suitable for using in animals and lack of a Pik3c3-deficient animal model preclude the possibility of conclusively defining a role for Pik3c3 in CNH in previous studies. Here, we report that insertion of an Frt-flanked PGK-Neo cassette into intron 19 of the mouse Pik3c3 gene resulted in a hypomorphic allele. This allowed us to create a unique mouse model and provide the first definitive genetic evidence demonstrating whether Pik3c3 is essential for the regulation of CNH. Our results indicate that homozygous Pik3c3 hypomorphic (Pik3c3Hypo/Hypo) mice express significantly low levels of Pik3c3 than heterozygous Pik3c3 hypomorphic (Pik3c3Hypo/WT) littermates, which already express a lower level of Pik3c3 than wild-type (Pik3c3WT/WT) littermates. Interestingly, after unilateral nephrectomy (UNX), Pik3c3Hypo/Hypo mice develop a significantly lower degree of CNH than Pik3c3WT/WT mice and Pik3c3Hypo/WT mice, as revealed by measurement of kidney weight, kidney-to-body weight ratio, renal protein-to-DNA ratio, and morphometric analysis of proximal tubular and glomerular size. Mechanistically, UNX-induced mammalian target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) in the remaining kidney was markedly inhibited in Pik3c3 hypomorphic mice. In conclusion, the present study reports a Pik3c3 hypomorphic mouse model and provides the first definitive evidence that Pik3c3 controls the degree of compensatory nephron hypertrophy. In addition, our signaling data provide the first definitive in vivo proof that Pik3c3 functions upstream of the mTORC1-S6 kinase 1-rpS6 pathway in the regulation of compensatory nephron hypertrophy.

mTORC1 Overactivation Leads to Abnormalities in Skeletal Development.

The mechanistic/mammalian target of rapamycin complex-1 (mTORC1) integrates multiple signaling pathways and regulates various cellular processes. Tuberous sclerosis complex 1 (Tsc1) and complex 2 (Tsc2) are critical negative regulators of mTORC1. Mouse genetic studies, including ours, have revealed that inactivation of mTORC1 in undifferentiated mesenchymal cells and chondrocytes leads to severe skeletal abnormalities, indicating a pivotal role for mTORC1 in skeletogenesis. Here, we show that hyperactivation of mTORC1 influences skeletal development through its expression in undifferentiated mesenchymal cells at the embryonic stage. Inactivation of Tsc1 in undifferentiated mesenchymal cells by paired-related homeobox 1 (Prx1)-Cre-mediated recombination led to skeletal abnormalities in appendicular skeletons. In contrast, Tsc1 deletion in chondrocytes using collagen type II α1 (Col2a1)-Cre or in osteoprogenitors using Osterix (Osx)-Cre did not result in skeletal defects in either appendicular or axial skeletons. These findings indicate that Tsc complex-mediated chronic overactivation of mTORC1 influences skeletal development at the embryonic stage through its expression in undifferentiated mesenchymal cells but not in chondrocytes or osteoprogenitors.

mTORC1 signaling is essential for neurofibromatosis type I gene modulated osteogenic differentiation of BMSCs.

Neurofibromatosis type I (NF1), which is caused by mutations in the NF1 gene, is a common autosomal dominant genetic disease leading to skeletal abnormalities. Both NF1 gene and mammalian target of rapamycin complex 1 (mTORC1) signaling are associated with the osteogenic differentiation of bone marrow stem cells (BMSCs). In this study, we hypothesized that mTORC1 signaling is involved in NF1-modulated osteoblast differentiation of BMSCs. Human BMSCs were cultured in an osteogenic induction medium. The expression of NF1 was either inhibited or overexpressed by transfecting NF1 with a specific small interfering RNA (siRNA) or pcDNA3.0 plasmid, respectively. In addition, an mTORC1 signaling inhibitor and agonist were used to investigate the effects of mTORC1 on NF1-modulated osteogenic differentiation of BMSCs. The results indicated that inhibiting the expression of NF1 with siRNA significantly decreased the mRNA levels of NF1, whereas overexpressing the expression of NF1 with pcDNA3.0 plasmid significantly increased the mRNA levels of NF1 at days 3, 7, 14 and 21 after culture. We observed reduced osteogenic differentiation and cell proliferation in the NF1-siRNA group and enhanced osteogenic differentiation and cell proliferation of BMSCs in the NF1-pcDNA3.0 group. The activity of mTORC1 signaling (p-mTORC1, p-S6K1, and p-4EBP1) was significantly upregulated in the NF1-siRNA group and significantly inhibited in the NF1-pcDNA3.0 group, 7 and 14 days after culture. The effects of NF1-siRNA and NF1-pcDNA3.0 on osteogenic differentiation of BMSCs and cell proliferation were reversed by mTORC1 inhibitor and agonist, respectively. In conclusion, NF1 modulates osteogenic differentiation and cell proliferation of human BMSCs and mTORC1 signaling is essential for this process.

mTORC1/rpS6 signaling complex modifies BTB transport function: an in vivo study using the adjudin model.

Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.

Maternal L-proline supplementation enhances fetal survival, placental development, and nutrient transport in mice†.

L-Proline (proline) in amniotic fluid was markedly increased during pregnancy in both pigs and sheep. However, in vivo data to support a beneficial effect of proline on fetal survival are not available. In this study, pregnant C57BL/6J mice were fed a purified diet supplemented with or without 0.50% proline from embryonic day 0.5 (E0.5) to E12.5 or term. Results indicated that dietary supplementation with proline to gestating mice enhanced fetal survival, reproductive performance, the concentrations of proline, arginine, aspartic acid, and tryptophan in plasma and amniotic fluid, while decreasing the concentrations of ammonia and urea in plasma and amniotic fluid. Placental mRNA levels for amino acid transporters, including Slc36a4, Slc38a2, Slc38a4, Slc6a14, and Na+/K+ ATPase subunit-1α (Atp1a1), fatty acid transporter Slc27a4, and glucose transporters Slc2a1 and Slc2a3, were augmented in proline-supplemented mice, compared with the control group. Histological analysis showed that proline supplementation enhanced labyrinth zone in the placenta of mice at E12.5, mRNA levels for Vegf, Vegfr, Nos2, and Nos3, compared with the controls. Western blot analysis showed that proline supplementation increased protein abundances of phosphorylated (p)-mTORC1, p-ribosomal protein S6 kinase (p70S6K), and p-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), as well as the protein level of GCN2 (a negative regulator of mTORC1 signaling). Collectively, our results indicate a novel functional role of proline in improving placental development and fetal survival by enhancing placental nutrient transport, angiogenesis, and protein synthesis.

Fibroblast growth factor receptor promotes progression of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer striking 200 000 Americans annually. Further, in organ transplant patients, there is a 65- to 100-fold increased incidence of cSCC compared to the general population. Excision of cSCC of the head and neck results in significant facial disfigurement. Therefore, increased understanding of the mechanisms involved in the pathogeneses of cSCC could identify means to prevent, inhibit, and reverse this process. In our previous studies, inhibition of fibroblast growth factor receptor (FGFR) significantly decreased ultraviolet B-induced epidermal hyperplasia and hyperproliferation in SKH-1 mice, suggesting an important role for FGFR signaling in skin cancer development. However, the role of FGFR signaling in the progression of cSCC is not yet elucidated. Analysis of the expression of FGFR in cSCC cells and normal epidermal keratinocytes revealed protein overexpression and increased FGFR2 activation in cSCC cells compared to normal keratinocytes. Further, tumor cell-specific overexpression of FGFR2 was detected in human cSCCs, whereas the expression of FGFR2 was low in premalignant lesions and normal skin. Pretreatment with the pan-FGFR inhibitor; AZD4547 significantly decreased cSCC cell-cycle traverse, proliferation, migration, and motility. Interestingly, AZD4547 also significantly downregulated mammalian target of rapamycin complex 1 and AKT activation in cSCC cells, suggesting an important role of these signaling pathways in FGFR-mediated effects. To further bolster the in vitro studies, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with SCC12A tumor xenografts treated with AZD4547 (15 mg/kg/bw, twice weekly oral gavage) exhibited significantly decreased tumor volume compared to the vehicle-only treatment group. The current studies provide mechanistic evidence for the role of FGFR and selectively FGFR2 in the early progression of cSCC and identifies FGFR as a putative therapeutic target in the treatment of skin cancer.

l-Leucine influx through Slc7a5 regulates inflammatory responses of human B cells via mammalian target of rapamycin complex 1 signaling.

Objectives: Increasing evidence has revealed the close correlation between immune cell functions and their intracellular metabolism. Mammalian target of rapamycin complex 1 (mTORC1) is the important metabolism-modulating signal that regulates cellular activities. In certain types of cell, it is known that mTORC1 activation depends on influx of l-leucine through an amino acid transporter, Slc7a5. In B cells, however, the expression and the role of Slc7a5 have never been investigated. Methods: CD19+ B cells were obtained from peripheral blood of healthy adults and stimulated by a toll-like receptor 9 ligand, CpG oligodeoxynucleotides. The expression of Slc7a5 and l-leucine uptake were evaluated by RT-PCR, flow cytometry and radioisotope assay. Then the effect of Slc7a5 inhibition on mTORC1 activity, plasmablast differentiation and production of IgG and inflammatory cytokines were analyzed. Results: CpG stimulation significantly induced the expression of Slc7a5 in B cells, resulting in l-leucine influx. Furthermore, inhibition of Slc7a5 abrogated mTORC1 activation, plasmablast differentiation, and production of IgG and inflammatory cytokines in CpG-stimulated B cells. Conclusion: l-leucine influx through Slc7a5 critically regulates mTORC1 activity and the immunological responses of human B cells. Slc7a5-mTORC1 pathway may provide a novel therapeutic strategy for autoimmune diseases.