Activation of activin type IB receptor signals in pancreatic β cells leads to defective insulin secretion through the attenuation of ATP-sensitive K channel activity

In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K⁺ channels (K_ATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of K_ATP channel activity.     

Induction of alkaline phosphatase and the glycoprotein hormone α subunit in HeLa cells by inhibitors of DNA polymerase

Levels of the glycoprotein hormone α subunit and alkaline phosphatase activity were increased in cultures of HeLa S₃ cells exposed to aphidicolin (0.2–10 μg/ml) or phosphonoformic acid (0.1–3 mm), inhibitors of DNA polymerase α. Induction was dependent on both the concentration and duration of exposure to the inhibitors and was prevented by cycloheximide and actinomycin D. Limited characterization of the induced α subunit and alkaline phosphatase activity suggest that they are similar to the uninduced proteins expressed by this cell line. Induction of both proteins by aphidicolin and phosphonoformic acid was enhanced by the simultaneous addition of 3 mm sodium butyrate but was depressed by 1 mm hydroxy urea. In contrast, both butyrate and hydroxy urea cause induction of these proteins when added alone to HeLa cultures. It is unlikely that a direct relationship exists between protein induction and the inhibition of DNA synthesis produced by aphidicolin and phosphonoformic acid since the concentrations required to produce half-maximal induction are 5 to 10 times greater than those needed to inhibit replication by 50%.     

Role of neuregulin-1β in dexamethasone-enhanced surfactant synthesis in fetal type II cells

It is well established that glucocorticoids elevate the production of fibroblast-pneumocyte factor (FPF), which induces type II cells to synthesize surfactant phospholipids. FPF, however, has not been identified and it is not clear whether it is a single factor or a complex mixture of factors. In this study it has been shown that, when lung fibroblasts are exposed to dexamethasone, the concentration of neuregulin-1β (NRG1β) in conditioned medium is elevated 2-fold (P < 0.05), even though NRG1β gene expression is unaffected. This, together with the finding that exposure of type II cells to NRG1β directly stimulates by 3-fold the rate of phospholipid synthesis (P < 0.05), suggests that NRG1β is a component of FPF that promotes lung development.     

α-Thrombin inhibits DNA synthesis in rat hepatocytes but not in hepatoma cells by receptor activation and proteolysis

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His₆ tag (rBtaPAP1_6H) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP1_6H had a specific activity of 3633 units mg⁻¹. SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of ∼24 kDa, which is in good agreement with previously reported data on PAP1. The K _m and k _cat values obtained for rBtaPAP1_6H were 59 μM and 3.5 s⁻¹, respectively. The optimum pH for activity was 9.0–9.5 and the optimum temperature was 37 °C. rBtaPAP1_6H was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH₂ (TRH), pGlu-Ala and pGlu-Val revealed K _i values of 44.1, 141 and 652.17 μM, respectively. The lowest K _i, observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP1_6H has a higher affinity for tripeptides over dipeptides.     

Isolation of an Arabidopsis thaliana casein kinase II β subunit by complementation in Saccharomyces cerevisiae

Casein kinase II is thought to play an essential role in the control of cell division and differentiation in all eukaryotes. Through complementation of a defective casein kinase II catalytic subunit gene from Saccharomyces cerevisiae, we isolated an Arabidopsis thaliana casein kinase II regulatory subunit homologue, CKB1. A second regulatory subunit was identified by low-stringency hybridization with CKB1.Casein kinase II from S. cerevisiae is composed of two catalytic () and two regulatory () subunits. Simultaneous disruption of the genes for the and subunits, CKA1 and CKA2, respectively, is lethal. Strain YDH8 has disruptions of CKA1 and CKA2; its viability depends on a temperature-sensitive allele of CKA2, cka2–8, carried on a centromeric plasmid. We screened an A. thaliana cDNA library, whose inserts are under the control of the galactose-inducible GAL10 promoter, for cDNAs which enabled YDH8 cells to grow at the restrictive temperature. One cDNA, CKB1, was isolated by this screen which had homology to cDNAs of casein kinase II subunits. A second cDNA, CKB2, was isolated by hybridization and was also able to suppress the YDH8 mutant phenotype.The proteins encoded by CKB1 and CKB2 are 80% identical. The carboxy-terminal two thirds of both proteins is ca. 54% identical to the regulatory subunits of casein kinase II from other species. The amino termini are unrelated to any other known proteins. CKB1 and CKB2 lack the conserved autophosphorylation site characteristic of animal subunits, but have potential casein kinase II phosphorylation sites in the same region. Suppression of the cka1 cka2–8 mutant phenotype occurs by interaction of CKB1 with the defective, cka2–8-encoded, catalytic subunit. Cells with disruptions in CKA1 and CKA2 are not rescued by expression of CKB1.     

Silencing of lncRNA ZFAS1 inhibits malignancies by blocking Wnt/β-catenin signaling in gastric cancer cells

Gastric cancer is a common malignancy with high mortality. Long noncoding RNA (lncRNA) zinc finger antisense (ZFAS)1 is upregulated in gastric cancer specimens compared with the para-carcinoma tissues. The silencing of ZFAS1 inhibited the growth, proliferation, cell cycle progress, migration, invasion and epithelial-mesenchymal transition (EMT), and enhanced the sensitivity to cis-platinum or paclitaxel in SGC7901 cells, as evidenced by the expression changes of proliferating cell nuclear antigen, Cyclin D1, Cyclin E, Cyclin B1, E-cadherin, N-cadherin, vimentin, matrix metalloproteinase (MMP)-2 and MMP-14. The ZFAS1 also activated the Wnt/β-catenin signaling. Subsequently, the ZFAS1 knockdown-induced the inhibition of migration, invasion, EMT and resistance to chemotherapeutic reagens was reversed by the overexpression of β-catenin. In summary, the silencing of ZFAS1 inhibited the growth, proliferation, cell cycle progress, migration, invasion, EMT and chemotherapeutic tolerance by blocking the Wnt/β-catenin signaling in gastric cancer cells.     

From insulin synthesis to secretion: Alternative splicing of type 2 ryanodine receptor gene is essential for insulin secretion in pancreatic β cells

Increases in the intracellular Ca²⁺ concentration in pancreatic islets, resulting from the Ca²⁺ mobilization from the intracellular source through the ryanodine receptor, are essential for insulin secretion by glucose. Cyclic ADP-ribose, a potent Ca²⁺ mobilizing second messenger synthesized from NAD⁺ by CD38, regulates the opening of ryanodine receptor. A novel ryanodine receptor mRNA (the islet-type ryanodine receptor) was found to be generated from the type 2 ryanodine receptor gene by the alternative splicing of exons 4 and 75. The islet-type ryanodine receptor mRNA is expressed in a variety of tissues such as pancreatic islets, cerebrum, cerebellum, and other neuro-endocrine cells, whereas the authentic type 2 ryanodine receptor mRNA (the heart-type ryanodine receptor) was found to be generated using GG/AG splicing of intron 75 and is expressed in the heart and the blood vessel. The islet-type ryanodine receptor caused a greater increase in the Ca²⁺ release by caffeine when expressed in HEK293 cells pre-treated with cyclic ADP-ribose, suggesting that the novel ryanodine receptor is an intracellular target for the CD38-cyclic ADP-ribose signal system in mammalian cells and that the tissue-specific alternative splicing of type 2 ryanodine receptor mRNA plays an important role in the functioning of the cyclic ADP-ribose-sensitive Ca²⁺ release.     

1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase

Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)₂D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)₂D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48% ± 5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)₂D-mediated regulation and hypothesized that 1,25(OH)₂D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)₂D treatment at 2 (73% ± 6 relative to vehicle) and 5 (56% ± 8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54% ± 12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)₂D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)₂D regulates lipid metabolism in malignant breast epithelial cells.     

Neuroendocrine control of follicle-stimulating hormone (FSH) secretion: II. Is follistatin-induced suppression of FSH secretion mediated via changes in activin availability and does it involve changes in gonadotropin-releasing hormone secretion?

The objective of the present study was to determine to what extent activin participates in setting the level of FSH secretion and if this regulation includes mediation via changes in GnRH secretion. We administered follistatin, the high-affinity binding protein for activin, to five ovariectomized sheep; we reasoned that the resultant binding of follistatin to activin should lower activin bioavailability and FSH secretion. Hypophyseal portal and peripheral blood samples were collected simultaneously at 10-min intervals for 18 h to measure GnRH, LH, FSH, and both activin-free and total follistatin. Six hours into collection, each ewe received 150 microg/kg i.v. of recombinant human follistatin-288. A week later, the same ewes were subjected to a second series of blood collections of similar length (time control). The FSH levels in pituitary portal blood were approximately 8-fold higher than those in the peripheral circulation. The FSH secretory patterns changed minimally during the time-control period. In contrast, follistatin had profound suppressive effects on FSH secretion. Maximal FSH suppression after FS-288 administration occurred at 5-6 h in the pituitary portal (65% suppression) and 9-10 h in the peripheral (48% suppression) circulation. Follistatin had no effect on GnRH or LH secretory patterns. Disappearance of total follistatin (i.e., free follistatin plus activin-bound follistatin) from the circulation was slower (P < 0.05) than that of free follistatin alone, suggesting that some of the follistatin was complexed with circulating activin, thus reducing the bioavailability of activin. The slower clearance of total follistatin and the lack of follistatin effects on GnRH secretion suggest that changes in activin bioavailability dictate the level of pituitary FSH secretion and that this is a pituitary-specific effect.     

TGF-β sensitivity is determined by N-linked glycosylation of the type II TGF-β receptor

N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability and localization, ligand-receptor binding and intracellular signalling. TβRII [type II TGF-β (transforming growth factor β) receptor] plays a crucial role in the TGF-β signalling pathway. Although N-linked glycosylation of TβRII was first demonstrated over a decade ago, it was unclear how this modification influenced TβRII biology. In the present study, we show that inhibiting the N-linked glycosylation process successfully hinders binding of TGF-β1 to TβRII and subsequently renders cells resistant to TGF-β signalling. The lung cancer cell line A549, the gastric carcinoma cell line MKN1 and the immortal cell line HEK (human embryonic kidney)-293 exhibit reduced TGF-β signalling when either treated with two inhibitors, including tunicamycin (a potent N-linked glycosylation inhibitor) and kifunensine [an inhibitor of ER (endoplasmic reticulum) and Golgi mannosidase I family members], or introduced with a non-glycosylated mutant version of TβRII. We demonstrate that defective N-linked glycosylation prevents TβRII proteins from being transported to the cell surface. Moreover, we clearly show that not only the complex type, but also a high-mannose type, of TβRII can be localized on the cell surface. Collectively, these findings demonstrate that N-linked glycosylation is essentially required for the successful cell surface transportation of TβRII, suggesting a novel mechanism by which the TGF-β sensitivity can be regulated by N-linked glycosylation levels of TβRII.     

GABAA receptor subunit β1 is involved in the formation of protease-resistant prion protein in prion-infected neuroblastoma cells

γ-Aminobutyric acid type A (GABAA) receptor β1 (gabrb1), a subunit of GABAA receptors involved in inhibitory effects on neurotransmission, was found to associate with the formation of protease-resistant prion protein in prion-infected neuroblastoma cells. Silencing of gabrb1 gene expression significantly decreased the abnormal prion protein level but paradoxically increased the normal prion protein level. Treatment with a gabrb1-specific inhibitor, salicylidene salicylhydrazide, dose-dependently decreased the abnormal prion protein level, but silencing of other GABAA receptor subunits’ gene expression and treatments with the receptor antagonists and agonists did not. Therefore, gabrb1 involvement in abnormal prion protein formation is independent of GABAA receptors.     

Losartan inhibits endothelial-to-mesenchymal transformation in mitral valve endothelial cells by blocking transforming growth factor-β-induced phosphorylation of ERK

Adult cardiac valve endothelial cells (VEC) undergo endothelial to mesenchymal transformation (EndMT) in response to transforming growth factor-β (TGFβ). EndMT has been proposed as a mechanism to replenish interstitial cells that reside within the leaflets and further, as an adaptive response that increases the size of mitral valve leaflets after myocardial infarction. To better understand valvular EndMT, we investigated TGFβ-induced signaling in mitral VEC, and carotid artery endothelial cells (CAEC) as a control. Expression of EndMT target genes α-smooth muscle actin (α-SMA), Snai1, Slug, and MMP-2 were used to monitor EndMT. We show that TGFβ-induced EndMT increases phosphorylation of ERK (p-ERK), and this is blocked by Losartan, an FDA-approved antagonist of the angiotensin II type 1 receptor (AT1), that is known to indirectly inhibit phosphorylation of ERK (p-ERK). Blocking TGF-β-induced p-ERK directly with the MEK1/2 inhibitor RDEA119 was sufficient to prevent EndMT. In mitral VECs, TGFβ had only modest effects on phosphorylation of the canonical TGF-β signaling mediator mothers against decapentaplegic homolog 3 (SMAD3). These results indicate a predominance of the non-canonical p-ERK pathway in TGFβ-mediated EndMT in mitral VECs. AT1 and angiotensin II type 2 (AT2) were detected in mitral VEC, and high concentrations of angiotensin II (AngII) stimulated EndMT, which was blocked by Losartan. The ability of Losartan or MEK1/2 inhibitors to block EndMT suggests these drugs may be useful in manipulating EndMT to prevent excessive growth and fibrosis that occurs in the leaflets after myocardial infarction.     

Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F₁ with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F₁ sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β_DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β_E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.