Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells

Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28 days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28 days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p < 0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47 ± 1.57% vs. 33.47 ± 1.34%, p < 0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway.     

Phosphoinositide 3-kinase/protein kinase B inhibition restores regulatory T cell's function in pulmonary sarcoidosis

Sarcoidosis is a systemic granulomatous disease associated with Th1/ regulatory T cells (Treg) paradigm. PI3K/Akt signaling, critical for maintaining Treg's homeostasis, is aberrantly activated in sarcoidosis patients. Here we tested the role of the PI3K inhibitors, LY294002 and BKM120, in immune modulation in experimental pulmonary sarcoidosis, concerning Th1/Th17/Treg immune profile detected by fluorescence-activated cell sorting analysis or quantitative polymerase chain reaction, as well as the effect on Treg's suppressive functions. Our investigation showed abnormal activation of PI3K/Akt signaling both in lung and Treg in pulmonary sarcoidosis, along with decreased frequency and damaged function of Treg. Blockage of PI3K suppressed this signaling in Treg, rebalanced Th1/Treg, inhibited the production of inflammatory cytokines, and enhanced Treg's function. These results demonstrate the key role of the PI3K/Akt signaling in regulating Th1/Th2 rebalances and indicates that PI3K/Akt signaling is critical for the optimal Treg responses in pulmonary sarcoidosis. Thus, PI3K inhibitors have potential for therapeutic translation, and can be candidate for add-on drugs to treat pulmonary sarcoidosis.     

Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.     

Leukotriene receptor antagonists,LY293111 and ONO-1078, protect neurons from the sPLA2-IB-induced neuronal cell death independently of blocking their receptors

In the ischemic brain, leukotrienes (LTs) are increased and their receptor antagonists protect neurons. However, it has not yet been sufficiently clarified how antagonists for LT receptors exhibit neuroprotective effects. In the present study, we evaluated protective effects of receptor antagonists for LTB₄ (LY293111) and cysteinyl LTs (ONO-1078) in the primary culture of rat cortical neurons. The group IB secretory phospholipase A₂ (sPLA₂-IB)-induced neuronal cell death had been established as the in vitro model for cerebral ischemia. sPLA₂-IB triggered the influx of Ca²⁺ into neurons via L-type voltage-dependent calcium channel (L-VDCC). Subsequently, the enzyme produced eicosanoids including LTB₄ before neuronal cell death. Neither administration of LTB₄ nor cysteinyl LTs such as LTC₄, LTD₄ and LTE₄ killed neurons. However, both LY293111 and ONO-1078 significantly prevented neurons from the neurotoxicity of sPLA₂-IB, suggesting that the two LT receptor blockers protected neurons through alternative pathways beside LT receptors. An L-VDCC blocker does not only inhibit the influx of Ca²⁺ into neurons but also rescues neurons from the sPLA₂-IB-induced neuronal cell death. The two LT receptor antagonists also blocked the sPLA₂-IB-induced Ca²⁺ influx significantly. Thus, LTs exhibited no neurotoxicity, but their receptor antagonists protected neurons directly in the in vitro ischemic model. Furthermore, the suppression of L-VDCC appeared to be involved in the neuroprotective effects of LY293111 and ONO-1078 independent of blocking their receptors.     

Conformational changes in tubulin upon binding cryptophycin-52 reveal its mechanism of action

Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC₅₀ values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we have determined the binding site of Cp-52, and its parent compound, cryptophycin-1, on HeLa tubulin, to a resolution of 3.3 Å and 3.4 Å, respectively, by cryo-EM and characterized this binding further by molecular dynamics simulations. The binding site was determined to be located at the tubulin interdimer interface and partially overlap that of maytansine, another cytotoxic tubulin inhibitor. Binding induces curvature both within and between tubulin dimers that is incompatible with the microtubule lattice. Conformational changes occur in both α-tubulin and β-tubulin, particularly in helices H8 and H10, with distinct differences between α and β monomers and between Cp-52-bound and cryptophycin-1-bound tubulin. From these results, we have determined: (i) the mechanism of action of inhibition of both microtubule polymerization and depolymerization, (ii) how the affinity of Cp-52 for tubulin may be enhanced, and (iii) where linkers for targeted delivery can be optimally attached to this molecule.     

Larazotide acetate promotes tight junction assembly in epithelial cells

Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in "leaky" Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins.     

The phosphoinositide 3-kinase inhibitor LY294002, decreases aminoacyl-tRNA synthetases, chaperones and glycolytic enzymes in human HT-29 colorectal cancer cells

The proposed anticancer drug LY294002, inhibits phosphoinositide-3 kinase (PI3K) that initiates a signalling pathway often activated in colorectal cancer (CRC). The effects of LY294002 (10 μM, 48 h) on the cytosolic, mitochondrial and nuclear proteomes of human HT-29 CRC cells have been determined using iTRAQ (isobaric tag for relative and absolute quantitation) and tandem mass spectrometry (MS/MS). Analysis of cells treated with LY294002 identified 26 differentially abundant proteins that indicate several mechanisms of action. The majority of protein changes were directly or indirectly associated with Myc and TNF-α, previously implicated in CRC progression. LY294002 decreased the levels of 6 aminoacyl-tRNA synthetases (average 0.39-fold) required for protein translation, 5 glycolytic enzymes (average 0.37-fold) required for ATP synthesis, and 3 chaperones required for protein folding. There was a 3.2-fold increase in lysozyme C involved in protein-glycoside hydrolysis. LY294002 increased cytosolic p53 with a concomitant decrease in nuclear p53, suggesting transfer of p53 to the cytosol where apoptosis might be initiated via the intrinsic mitochondrial pathway. Protein changes described here suggest that the anti-angiogenic effects of LY294002 may be related to p53; the mutational status of p53 in CRC may be an important determinant of the efficacy of PI3K inhibitors for treatment.     

5,10-Methenyltetrahydrofolate synthetase activity is increased in tumors and modifies the efficacy of antipurine LY309887

Methenyltetrahydrofolate synthetase (MTHFS) expression enhances folate-dependent de novo purine biosynthesis. In this study, the effect of increased MTHFS expression on the efficacy of the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor LY309887 was investigated in SH-SY5Y neuroblastoma. GARFT catalyzes the incorporation of formate, in the form of 10-formyltetrahydrofolate, into the C8 position of the purine ring during de novo purine biosynthesis. SH-SY5Y neuroblastoma with increased MTHFS expression displayed a 4-fold resistance to the GARFT inhibitor LY309887, but did not exhibit resistance to the thymidylate synthase inhibitor Pemetrexed. This finding supports a mechanism whereby MTHFS increases the availability of 10-formyltetrahydrofolate for GARFT. MTHFS expression is elevated in animal tumor tissues compared to surrounding normal tissue, consistent with the dependence of transformed cells on de novo purine biosynthesis. The level of MTHFS expression in tumors may predict the efficacy of antipurine agents that target GARFT.     

Epinephrine-induced hyperpolarization of pancreatic islet cells is sensitive to PI3K-PDK1 signaling

Epinephrine inhibits insulin release by activation of K⁺ channels and subsequent hyperpolarization of pancreatic beta cells. The present study explored whether epinephrine-induced hyperpolarization is modified by phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositide-dependent kinase PDK1. Perforated patch-clamp was performed in islet cells isolated from PDK1 hypomorphic mice (pdk1^fl/fl), expressing only 20% of PDK1, and in their wild-type littermates. At 16.8 mM glucose, the cell membrane was hyperpolarized by epinephrine (1 μM), an effect significantly blunted in pdk1^fl/fl and abrogated in wild-type cells by inhibition of PI3K with wortmannin (100 nM) or LY294002 (10 μM). The hyperpolarizing effect of epinephrine in pancreatic islet cells is thus sensitive to PI3K and PDK1.     

Cell migration and activated PI3K/AKT-directed elongation in the developing rat Müllerian duct

In vertebrates, the Müllerian duct elongates along the Wolffian duct, a mesonephric structure that is required for Müllerian duct formation. Recently, several genes required for initial Müllerian duct formation have been identified. However, the precise mechanism of Müllerian duct elongation remains to be elucidated. In this study, we investigated dynamic morphological changes in the elongating Müllerian duct in rat urogenital ridges in organ culture manipulated by microincision and/or chemical inhibitors. Mechanical division of the developing Müllerian duct showed that epithelial cells of the Müllerian duct actively migrate along the anterior-posterior axis independent of the proliferative expansion of the anterior portion of the duct. We found that the PI3K/AKT signaling pathway is activated in the Müllerian duct epithelium and is required for elongation of the tip of the duct; however, migration of Müllerian duct epithelial cells proximal to the tip remains intact when PI3K/AKT is inactivated. Although much is known about the molecular and cellular mechanisms leading to Müllerian duct regression, the present findings provide a fuller understanding of the mechanisms contributing to Müllerian duct formation and to the general process of early tubulogenesis.