LncRNA RP6-65G23.1 accelerates proliferation and inhibits apoptosis via p-ERK1/2/p-AKT signaling pathway on keratinocytes

Long non-coding RNAs (LncRNAs) play essential roles in the development of various diseases including hepatic carcinoma, melanoma, and psoriasis. Meanwhile, lncRNA-RP6-65G23.1 was upregulated in psoriasis. However, it is still unclear whether lncRNA-RP6-65G23.1 expression is upregulated and contributes to keratinocytes proliferation and apoptosis, and which mechanisms are responsible for these processes. The aims of this study are to address these issues. RP6-65G23.1 was significantly upregulated in M5-stimulated keratinocytes and stimulated the proliferation and inhibited the apoptosis of HaCaT cells. Knockdown of RP6-65G23.1 resulted in defects of growth and increased rates of apoptosis in HaCaT cells, while overexpression of RP6-65G23.1 manifested the opposite effects. Consistently, the expression of antiapoptotic proteins Bcl-xl and Bcl2 were decreased in RP6-65G23.1-knockdown cells but elevated in RP6-65G23.1 overexpression cells. In addition, RP6-65G23.1 depletion blunted the activity of extracellular regulated kinase 1/2 (ERK1/2) and AKT signaling pathways and induced G₁/S-growth arrest. By contrast, overexpression of RP6-65G23.1 activates the ERK1/2 and AKT signaling pathways and inhibits the expression of p21 and p27 in an AKT-dependent manner leading to promote the G1/S progression. Our results suggested that lncRNA-RP6-65G23.1 would contribute to the pathogenesis of psoriasis by regulating the proliferation and apoptosis of keratinocytes via the p-ERK1/2 and p-AKT pathways.     

mTORC1 signaling activation increases intestinal stem cell activity and promotes epithelial cell proliferation

The crypt-villus axis of the intestine undergoes a continuous renewal process that is driven by intestinal stem cells (ISCs). However, the homeostasis is disturbed under constant exposure to high ambient temperatures, and the precise mechanism is unclear. We found that both EdU⁺ and Ki67⁺ cell ratios were significantly reduced after exposure to 41°C, as well as the protein synthesis rate of IPEC-J2 cells, and the expression of ubiquitin and heat shock protein 60, 70, and 90 were significantly increased. Additionally, heat exposure decreased enteroid expansion and budding efficiency, as well as induced apoptosis after 48 hr; however, no significant difference was observed in the apoptosis ratio after 24 hr. In the process of heat exposure, the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway was significantly inhibited in both IPEC-J2 cells and enteroids. Correspondingly, treatment of IPEC-J2 and enteroids with the mTORC1 agonist MHY1485 at 41°C significantly attenuated the inhibition of proliferation and protein synthesis, increased the ISC activity, and promoted expansion and budding of enteroid. In summary, we conclude that the mTORC1 signaling pathway regulates intestinal epithelial cell and stem cell activity during heat exposure-induced injury.     

MiR-20a-3p regulates TGF-β1/Survivin pathway to affect keratinocytes proliferation and apoptosis by targeting SFMBT1 in vitro

Psoriasis is a common immune-mediated chronic inflammatory skin disease characterized by abnormal keratinocyte proliferation, differentiation and apoptosis. However, the exact etiology and pathogenesis are still unclear. Evidence is rapidly accumulating for the role of microRNAs in psoriasis. It has been demonstrated that Interleukin-22 (IL-22) plays vital role in T cell-mediated immune response by interacting with keratinocytes in the pathogenesis of psoriasis. The aim of our study was to explore the possible functional role of miR-20a-3p in psoriasis and in IL-22 induced keratinocyte proliferation. Here, we found that miR-20a-3p was down-regulated in psoriatic lesions and in HaCaT cells (human keratinocyte cell line) treated by IL-22 stimulation. Functional experiments showed that overexpression of miR-20a-3p in HaCaT cells suppressed proliferation and induced apoptosis while its knockdown promoted cell proliferation and reduces cell apoptosis. Mechanistically, SFMBT1 was identified as the direct target of miR-20a-3p by dual luciferase reporter assay. SFMBT1 knockdown was demonstrated to inhibit cell growth and induced apoptosis, which was consistent with the function of miR-20a-3p upregulation in HaCaT cells. In addition, results of western blot analysis showed that miR-20a-3p upregulation or SFMBT1 knockdown changed the protein expression levels of TGF-β1 and survivin. Our findings suggest that miR-20a-3p play roles through targeting SFMBT1 and TGF-β1/Survivin pathway in HaCaT cells, and loss of miR-20a-3p in psoriasis may contribute to hyperproliferation and aberrant apoptosis of keratinocytes.     

Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.     

Lycopene Protects Keratinocytes Against UVB Radiation‐Induced Carcinogenesis via Negative Regulation of FOXO3a Through the mTORC2/AKT Signaling Pathway

Lycopene, one of the most potent anti‐oxidants, has been reported to exhibit potent anti‐proliferative properties in a wide range of cancer cells through modulation of the cell cycle and apoptosis. Forkhead box O3 (FOXO3a) plays a pivotal role in modulating the expression of genes involved in cell death. Herein, we investigated the role of FOXO3a signaling in the anti‐cancer effects of lycopene. Results showed that lycopene pretreatment attenuated UVB‐induced cell hyper‐proliferation and promoted apoptosis, accompanied by decreased cyclin‐dependent kinase 2 (CDK2) and CDK4 complex in both human keratinocytes and SKH‐1 hairless mice. FOXO3a is phosphorylated in response to UVB irradiation and sequestered in the cytoplasm, while lycopene pretreatment rescued this sensitization. Gene ablation of FOXO3a attenuated lycopene‐induced decrease in cell hyper‐proliferation, CDK2, and CDK4 complex, indicating a critical role of FOXO3a in the lycopene‐induced anti‐proliferative effect of keratinocytes during UVB irradiation. Transfection with FOXO3a siRNA inhibited the lycopene‐induced increase in cell apoptosis, BAX and cleaved PARP expression. Moreover, loss of AKT induced further accelerated lycopene‐induced FOXO3a dephosphorylation, while loss of mechanistic target of rapamycin complex 2 (mTORC2) by transfection with RICTOR siRNA induced levels of AKT phosphorylation comparable to those obtained with lycopene. In contrast, overexpression of AKT or mTORC2 decreased the effects of lycopene on the expression of FOXO3a as well as AKT phosphorylation, suggesting that lycopene depends on the negative modulation of mTORC2/AKT signaling. Taken together, our findings demonstrate that the mTORC2/AKT/FOXO3a axis plays a critical role in the anti‐proliferative and pro‐apoptotic effects of lycopene in UVB‐induced photocarcinogenesis. J. Cell. Biochem. 119: 366–377, 2018. © 2017 Wiley Periodicals, Inc.     

Knockdown of long noncoding RNA urothelial carcinoma associated 1 inhibits colorectal cancer cell proliferation and promotes apoptosis via modulating autophagy

Long noncoding RNA urothelial carcinoma associated 1 (UCA1) has been implicated in the growth and metastasis of colorectal cancer (CRC), and autophagy contributes to tumorigenesis and cancer cell survival. However, the regulatory role of UCA1 in CRC cell viability by modulating autophagy remains unclear. In the present study, a significant positive correlation was observed between UCA1 and microtubule-associated protein 1 light chain 3 (LC3) levels, and the elevated UCA1 was negatively correlated with the PKB/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway in 293T cells. Downregulation of UCA1 inhibited autophagy activation and cell proliferation, whereas the apoptosis was increased and the cell cycle was arrested in G2 stage. The next results showed that UCA1 was markedly upregulated in Caco-2 cells. Knockdown of UCA1 significantly decreased the LC3-II and autophagy-related gene 5 (ATG5) protein levels and resulted in an increase in p62 expression. Conversely, the autophagy activator rapamycin (RAPA) reversed the effects. Furthermore, downregulated UCA1 decreased Caco-2 cells population in the G1 phase and increased the cells number in G2 phage. The cell proliferation was inhibited, and apoptosis rate was promoted. More important, RAPA could also abrogate the changes induced by knockdown of UCA1. Collectively, these data demonstrated that downregulated UCA1 induced autophagy inhibition, resulting in suppressing cell proliferation and promoting apoptosis, which suggested that UCA1 might serve as a potential new oncogene to regulate CRC cells viability by modulating autophagy.     

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.     

Calcitonin gene-related peptide regulates the expression of vascular endothelial growth factor in human HaCaT keratinocytes by activation of ERK1/2 MAPK

Psoriasis is a chronic disease characterized by abnormal epidermal proliferation, inflammation and angiogenesis. The pathogenetic process resulting in hypervascularity remains to be further investigated. It has been reported that a potent angiogenic factor, vascular endothelial growth factor (VEGF) is overexpressed in psoriatic epidermis and that the level of calcitonin gene-related peptide (CGRP) is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. We hypothesized that CGRP might regulate the expression of VEGF by human keratinocytes. VEGF expression in the CGRP-treated human keratinocytes was investigated and the CGRP signaling pathways were examined with respect to VEGF expression. The mRNA and protein levels of VEGF by CGRP were increased in a concentration-dependent manner. However, this increase was abrogated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor PD98059. The CGRP-mediated VEGF induction was also effectively inhibited by a pretreatment with the CGRP receptor antagonist CGRP 8-37. In addition, CGRP treatment induced rapid phosphorylation of ERK1/2, PD98059 and CGRP 8-37 were able to inhibit CGRP-induced ERK1/2 phosphorylation. These results suggest that CGRP regulates the expression of VEGF through the CGRP receptor and ERK1/2 MAPK signaling pathway in human HaCaT keratinocytes.     

GRB10 regulates β-cell mass by inhibiting β-cell proliferation and stimulating β-cell dedifferentiation

Decreased functional β-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1 cells. β-cell-specific knockout of Grb10 in mice increased β-cell mass and improved β-cell function. Grb10-deficient β-cells exhibit enhanced mTORC1 signaling and reduced β-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced β-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of β-cell dedifferentiation and β-cell mass, which exerts its effect by inhibiting mTORC1 signaling.     

mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1-JNK pathway

Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1-JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1-JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE-JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1-JNK pathway.     

Alpha-synuclein overexpression negatively regulates insulin receptor substrate 1 by activating mTORC1/S6K1 signaling

Alpha-synuclein (α-Syn) is a major component of Lewy bodies, a pathological feature of Parkinson's and other neurodegenerative diseases collectively known as synucleinopathies. Among the possible mechanisms of α-Syn-mediated neurotoxicity is interference with cytoprotective pathways such as insulin signaling. Insulin receptor substrate (IRS)-1 is a docking protein linking IRs to downstream signaling pathways such as phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K)1; the latter exerts negative feedback control on insulin signaling, which is impaired in Alzheimer's disease. Our previous study found that α-Syn overexpression can inhibit protein phosphatase (PP)2A activity, which is involved in the protective mechanism of insulin signaling. In this study, we found an increase in IRS-1 phosphorylation at Ser636 and decrease in tyrosine phosphorylation, which accelerated IRS-1 turnover and reduced insulin-Akt signaling in α-Syn-overexpressing SK-N-SH cells and transgenic mice. The mTOR complex (C)1/S6K1 blocker rapamycin inhibited the phosphorylation of IRS-1 at Ser636 in cells overexpressing α-Syn, suggesting that mTORC1/S6K1 activation by α-Syn causes feedback inhibition of insulin signaling via suppression of IRS-1 function. α-Syn overexpression also inhibited PP2A activity, while the PP2A agonist C2 ceramide suppressed both S6K1 activation and IRS-1 Ser636 phosphorylation upon α-Syn overexpression. Thus, α-Syn overexpression negatively regulated IRS-1 via mTORC1/S6K1 signaling while activation of PP2A reverses this process. These results provide evidence for a link between α-Syn and IRS-1 that may represent a novel mechanism for α-Syn-associated pathogenesis.     

Circ_0061012 contributes to IL-22-induced proliferation,migration and invasion in keratinocytes through miR-194-5p/GAB1 axis in psoriasis

Psoriasis is a chronic inflammation-associated skin disorder featured by excessive proliferation and abnormal differentiation of keratinocytes. Here, we intended to investigate the role of circular RNA 0061012 (circ_0061012) in psoriasis progression. The expression of circ_0061012, SLMO2-ATP5E readthrough (SLMO2-ATP5E) messenger RNA (mRNA), microRNA-194-5p (miR-194-5p) and GRB2 associated binding protein 1 (GAB1) mRNA was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and metastasis were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and transwell assays. Western blot assay was used to measure the protein levels of Ki67, matrix metallopeptidase 9 (MMP9) and GAB1. Dual-luciferase reporter assay and RNA immune co-precipitation (RIP) assay were used to verify the interaction between miR-194-5p and circ_0061012 or GAB1. Circ_0061012 abundance was significantly enhanced in lesional skin samples from psoriasis patients than that in normal skin specimens from healthy volunteers. Interleukin-22 (IL-22) treatment increased the expression of circ_0061012 in a dose-dependent manner. Circ_0061012 silencing alleviated IL-22-induced promoting effects in the proliferation, migration and invasion of HaCaT cells. Circ_0061012 interacted with miR-194-5p, and miR-194-5p knockdown counteracted circ_0061012 silencing-mediated influences in IL-22-induced HaCaT cells. GAB1 was a target of miR-194-5p in HaCaT cells, and miR-194-5p hampered proliferation and metastasis which were induced by IL-22 partly through targeting GAB1. Circ_0061012 elevated the expression of GAB1 through sponging miR-194-5p in HaCaT cells. Circ_0061012 accelerated IL-22-induced proliferation and metastasis in HaCaT cells through enhancing GAB1 expression via sponging miR-194-5p in psoriasis.     

Pharmacological inhibition of S6K1 impairs self‐renewal and osteogenic differentiation of bone marrow stromal cells

mTORC1 signaling not only plays important physiological roles in the regulation of proliferation and osteogenic differentiation of BMSCs, but also mediates exogenous Wnt‐induced protein anabolism and osteoblast differentiation. However, the downstream effectors of the mTORC1 signaling in the above processes are still poorly understood. In this study, we explored the specific role of S6K1, one of the major targets of the mTORC1 pathway, in BMSCs self ‐ renewal and osteogenic differentiation. We first found that S6K1 was active in primary mouse bone marrow stromal cells, and further activated upon osteogenic induction. We then determined the effects of S6K1 inhibition by LY2584702 Tosylate, a selective inhibitor of S6K1 (hereafter S6KI), using both primary mouse bone marrow stromal cells and ST2 cells. Colony‐Forming Unit‐Fibroblast (CFU‐F) assays showed that S6KI dramatically reduced the total number of colonies formed in primary BMSCs cultures. Under the basal osteogenic culture condition, S6KI significantly inhibited mRNA expression of osteoblast marker genes (Sp7, Bglap, Ibsp, and Col1a1), ALP activity and matrix mineralization. Upon Wnt3a treatments, S6KI inhibited Wnt3a‐induced osteoblast differentiation and expression of protein anabolism genes in ST2 cells, but to a much lesser degree than rapamycin (a specific inhibitor of mTORC1 signaling). Collectively, our findings have demonstrated that pharmacological inhibition of S6K1 impaired self ‐ renewal and osteogenic differentiation of BMSCs, but only partially suppressed exogenous Wnt3a‐induced osteoblast differentiation and protein anabolism.     

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.     

Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.