mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

Regulation of pancreatic β-cell function and mass dynamics by prostaglandin signaling

Prostaglandins (PGs) are signaling lipids derived from arachidonic acid (AA), which is metabolized by cyclooxygenase (COX)-1 or 2 and class-specific synthases to generate PGD₂, PGE₂, PGF_2α, PGI₂ (prostacyclin), and thromboxane A₂. PGs signal through G-protein coupled receptors (GPCRs) and are important modulators of an array of physiological functions, including systemic inflammation and insulin secretion from pancreatic islets. The role of PGs in β-cell function has been an active area of interest, beginning in the 1970s. Early studies demonstrated that PGE₂ inhibits glucose-stimulated insulin secretion (GSIS), although more recent studies have questioned this inhibitory action of PGE₂. The PGE₂ receptor EP3 and one of the G-proteins that couples to EP3, Gα_Z, have been identified as negative regulators of β-cell proliferation and survival. Conversely, PGI₂ and its receptor, IP, play a positive role in the β-cell by enhancing GSIS and preserving β-cell mass in response to the β-cell toxin streptozotocin (STZ). In comparison to PGE₂ and PGI₂, little is known about the function of the remaining PGs within islets. In this review, we discuss the roles of PGs, particularly PGE₂ and PGI₂, PG receptors, and downstream signaling events that alter β-cell function and regulation of β-cell mass.     

Interleukin-1α enhances the aggressive behavior of pancreatic cancer cells by regulating the α6β1-integrin and urokinase plasminogen activator receptor expression

Background  

In human pancreatic cancer progression, the α₆β₁-integrin is expressed on cancer cell surface during invasion and metastasis formation. In this study, we investigated whether interleukin (IL)-1α induces the alterations of integrin subunits and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) expression in pancreatic cancer cells. We hypothesize that the alterations of integrin subunits and uPA/uPAR expression make an important role in signaling pathways responsible for biological behavior of pancreatic cancer cells.     

Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells

The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.     

Synthesis of β-glucanase and other extracellular proteins by cells and protoplasts of the yeast Pichia polymorpha

The synthesis of β-glucanase either by cells or by protoplasts of the yeast Pichia polymorpha has been found to occur in the presence of 2-deoxy-d-glucose in the growth medium. On the other hand, the synthesis of typical extracellular proteins such as invertase and acid phosphatase is strongly affected by the presence of the drug. The degree of inhibition is, however, directly related to the 2-deoxy-d-glucose concentration.     

Conversion of pancreatic α cells into insulin-producing cells modulated by β-cell insufficiency and supplemental insulin administration

The emergence of bihormonal (BH) cells expressing insulin and glucagon has been reported under diabetic conditions in humans and mice. Whereas lineage tracing studies demonstrated that glucagon-producing α cells can be reprogrammed into BH cells, the underlying dynamics of the conversion process remain poorly understood. In the present study, we investigated the identities of pancreatic endocrine cells by genetic lineage tracing under diabetic conditions. When β-cell ablation was induced by alloxan (ALX), a time-dependent increase in BH cells was subsequently observed. Lineage tracing experiments demonstrated that BH cells originate from α cells, but not from β cells, in ALX-induced diabetic mice. Notably, supplemental insulin administration into diabetic mice resulted in a significant increase in α-cell-derived insulin-producing cells that did not express glucagon. Furthermore, lineage tracing in Ins2^Akita diabetic mice demonstrated a significant induction of α-to-β conversion. Thus, adult α cells have plasticity, which enables them to be reprogrammed into insulin-producing cells under diabetic conditions, and this can be modulated by supplemental insulin administration.     

PKCι regulates the expression of PDL1 through multiple pathways to modulate immune suppression of pancreatic cancer cells

To investigate the impact of oncogenic protein kinase C isoform ι (PKCι) on the microenvironment and the immunogenic properties of pancreatic tumors, we prohibit PKCι activity in various pancreatic ductal adenocarcinoma (PDAC) cell lines and co-culture them with human natural killer NK92 cells. The results demonstrate that PKCι suppression enhances the susceptibility of PDAC to NK cytotoxicity and promotes the degranulation and cytolytic activity of co-cultured NK92 cells. Mechanistic studies pinpoint that downstream of KRAS, both YAP1 and STAT3 are recruited by oncogenic PKCι to elevate the expression of PDL1, contributing to constitute an immune suppressive microenvironment in PDAC. Co-culture with NK92 further induces PDL1 upregulation via STAT3 to stimulate immune escape of PDAC cells. Subsequently, inhibition of PKCι in PDAC alleviates the immune suppression and enhances the cytotoxicity of NK92 towards PDAC through restraining PDL1 overexpression. Combined with PD1/PDL1 blocker, PKCι inhibitor remarkably elevates the cytotoxicity of NK92 against PDAC cells in vitro, establishing PKCι inhibitor as a promising candidate for boosting the immunotherapy of PDAC.     

Neopterin negatively regulates expression of ABCA1 and ABCG1 by the LXRα signaling pathway in THP-1 macrophage-derived foam cells

To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis.     

Tubulin-dependent secretion of S100A6 and cellular signaling pathways activated by S100A6-integrin β1 interaction

S100A6 is a calcium binding protein expressed mainly in fibroblasts and epithelial cells. Interestingly, S100A6 is also present in extracellular fluids. Recently we have shown that S100A6 is secreted by WJMS cells and binds to integrin β1 (Jurewicz et al., 2014). In this work we describe for the first time the mechanism of S100A6 secretion and signaling pathways activated by the S100A6-integrin β1 complex. We show that colchicine suppressed the release of S100A6 into the cell medium, which indicates that the protein might be secreted via a tubulin–dependent pathway. By applying double immunogold labeling and immunofluorescence staining we have shown that S100A6 associates with microtubules in WJMS cells. Furthermore, results obtained from immunoprecipitation and proximity ligation assay (PLA), and from in vitro assays, reveal that S100A6 is able to form complexes with α and β tubulin in these cells, and that the S100A6-tubulin interaction is direct. We have also found that the S100A6 protein, due to binding to integrin β1, activates integrin-linked kinase (ILK), focal adhesion kinase (FAK) and p21-activated kinase (PAK). Our results suggest that binding of S100A6 to integrin β1 affects cell adhesion/proliferation due to activation of ILK and FAK signaling pathways.     

shRNA targeting β1-integrin suppressed proliferative aspects and migratory properties of airway smooth muscle cells

Dysfunction of airway smooth muscle (ASM) is an essential feature of airway remodeling in chronic asthma. However, the precise mechanisms of this pathological process have not been well studied. In previous study, we found that β1-integrin, which was dramatically upregulated in ASM cells in an asthmatic mouse model, was associated with the cell proliferation. In this study, we employed short hairpin RNA (shRNA) targeting β1-integrin to assess the effect of down-regulation of this receptor on the proliferative aspects and migratory properties of ASM cells in vitro. The cells were treated with shRNA expression vectors directed against β1-integrin, control vectors that included the blank control, empty vector without shRNA, and mismatched shRNA, respectively. The mRNA and protein expressions of β1-integrin were determined by real-time PCR and Western blotting. Cell proliferation was measured by BrdU ELISA and cell cycle by fluorescence-activated cell sorter. Cell apoptosis was detected by Annexin V-PE/7-AAD staining. Cell migration assays were evaluated by transwell assay and expression of IL-6 and IL-8 by ELISA. The results revealed that shRNA targeting β1-integrin significantly decreased the mRNA and protein expressions of β1-integrin, enhanced the proportion of cells in G0/G1 phase, decreased the proportion in S phase, promoted cell apoptosis, inhibited cell proliferation, migration, IL-6 and IL-8 secretion in vitro. In conclusion, the overexpression of β1-integrin in ASM cells is essential for airway dysfunction development because it promotes proliferative aspects and migratory properties of ASM cells. Importantly, shRNA targeting β1-integrin may provide a new approach to preventing airway remodeling in chronic asthma.     

MiR-143-3p facilitates motility and invasiveness of endometriotic stromal cells by targeting VASH1/TGF-β signaling

Endometriosis is a benign gynecological disease. Accumulating evidence has revealed the participation of dysregulated miRNAs in the progression of endometriosis. Here, the function and molecular mechanism of miR-143?3p in endometriosis were investigated. The levels of vasohibin 1 (VASH1) and miR-143?3p in endometrial tissues and endometriotic stromal cells (ESCs) were detected by RT-qPCR. Migrative and invasive phenotypes of ESCs were tested by Transwell assays. The protein expression of VASH1, TGF-β signaling markers, and epithelial to mesenchymal transition (EMT) markers was examined by western blotting. The targeted relationship between miR-143?3p and VASH1 was confirmed by bioinformatics analysis and luciferase reporter assay. We found that miR-143?3p expression was significantly upregulated in ectopic endometrial tissues compared to that in eutopic and normal endometrial tissues. MiR-143?3p knockdown restrained EMT process, invasive and migrative behaviors of ESCs. Mechanically, miR-143?3p targeted VASH1 and negatively regulated VASH1. VASH1 downregulation reserved the effects of miR-143?3p knockdown in ESCs. MiR-143?3p activated TGF-β signaling via targeting VASH1. Furthermore, activation of TGF-β signaling counteracted the miR-143?3p knockdown-caused suppression of migration, invasion and EMT process in ESCs. Overall, miR-143?3p activates TGF-β signaling by targeting VASH1 to facilitate migration and invasion of ESCs.     

Localization of thymosin β10 in breast cancer cells: relationship to actin cytoskeletal remodeling and cell motility

Beta-thymosins are polypeptides involved in the regulation of actin polymerization and thymosin β10 and β4 have been implicated in sequestration of monomeric (G-) actin. Additionally, experimental overexpression of thymosin β10 has been found to result in increases in F-actin bundles as well as in cell motility and spreading. We have studied the distribution of endogenously expressed thymosin β10 in cultured human breast cancer cell lines. Both unperturbed monolayer cultures and wound-healing models were examined using double-staining for thymosin β10 and polymerized (F-) actin. Our findings show that thymosin β10 is expressed in all three-cancer cell lines (SK-BR-3, MCF-7 and MDA-MB-231) studied. No or little staining was detected in confluent cells, whereas strong staining occurred in semiconfluent cells and in cells populating monolayer wounds. Importantly, the distribution of staining for thymosin β10 was inverse of staining for F-actin. These data support a physiological role for thymosin β10 in sequestration of G-actin as well as in cancer cell motility.