Soybean β51–63 peptide stimulates cholecystokinin secretion via a calcium-sensing receptor in enteroendocrine STC-1 cells

We previously demonstrated that intraduodenal administration of an arginine-rich β51–63 peptide in soybean β-conglycinin suppresses food intake via cholecystokinin (CCK) secretion in rats. However, the cellular mechanisms by which the β51–63 peptide induces CCK secretion remain to be clarified. In the present study, we examined whether the extracellular calcium-sensing receptor (CaR) mediates β51–63-induced CCK secretion in murine CCK-producing enteroendocrine cell line STC-1. CCK secretion and changes in intracellular Ca²⁺ concentration in response to β51–63 peptide were measured in STC-1 cells under various extracellular Ca²⁺ concentrations and after treatment with a CaR antagonist. Intracellular Ca²⁺ concentrations in response to β51–63 peptide and extracellular Ca²⁺ were also measured in CaR-expressing human embryonic kidney (HEK-293) cells. The β51-63 peptide induced CCK secretion and intracellular Ca²⁺ mobilization in STC-1 cells under normal (1.2 mM) extracellular Ca²⁺ conditions in a dose-dependent manner. These responses to β51–63 peptide were reduced by the removal of intra- or extracellular Ca²⁺ but enhanced by increasing extracellular Ca²⁺ concentrations. Intracellular Ca²⁺ mobilization induced by extracellular Ca²⁺ was also increased by the pretreatment with β51–63 peptide. Treatment with a specific CaR antagonist (NPS2143) inhibited β51–63-induced CCK secretion and intracellular Ca²⁺ mobilization. In addition, HEK-293 cells transfected with CaR acquired sensitivity to the β51–63 peptide. From these results, we conclude that CaR is the β51–63 peptide sensor responsible for the stimulation of CCK secretion in enteroendocrine STC-1 cells.     

Soybean β-Conglycinin Bromelain Hydrolysate Stimulates Cholecystokinin Secretion by Enteroendocrine STC-1 Cells to Suppress the Appetite of Rats under Meal-Feeding Conditions

A peptic digest of soybean β-conglycinin (BconP) suppresses the appetite in rats through cholecystokinin (CCK) secretion by enteroendocrine cells. We investigate in this study more appetite-suppressing hydrolysates. β-Conglycinin hydrolyzed with food-processing proteases thermolysin (BconT), bromelain (BconB), chymotrypsin, protease S, and protease M was examined for CCK-secreting activity in a CCK-producing cell line for comparison with BconP. The potent CCK-releasing hydrolysates were then tested for their suppression of the food intake by rats. BconB, BconT, and BconP stimulated high CCK secretion, with the highest by BconB. Orogastric preloading by BconB, but not by BconT, suppressed the 60-min food intake. A meal-feeding trial twice a day in the morning (a.m.) and evening (p.m.) for 10 d showed that BconB preloading before every meal attenuated the p.m. meal size, but not that a.m., resulting in an overall reduction of the daily meal size. These results demonstrate that the bromelain hydrolysate of β-conglycinin having potent CCK-releasing activity suppressed the appetite of rats under meal-feeding conditions.     

Pneumocystis cell wall β-glucan stimulates calcium-dependent signaling of IL-8 secretion by human airway epithelial cells

Background

During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.

Method

Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.

Results

PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.

Conclusion

Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.     

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β-Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases. Cell-based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH-SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β-C-terminal fragment (β-CTF). Additionally, β-CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ-secretase-independent and proteasome-dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase-independent alteration of β-CTF levels.     

Inhibition of HIV-1 infection of peripheral blood mononuclear cells by a monoclonal antibody that binds to phosphoinositides and induces secretion of β-chemokines

A murine IgG mAb, WR321, selected for the ability to bind to phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, but an inability to bind to any of 17 other lipids, including phosphatidylinositol, was examined as a probe for studying interactions of HIV-1 with primary human peripheral blood mononuclear cells. The WR321 mAb broadly neutralized CCR5-tropic strains of HIV-1 to prevent infection of the cells. The mAb also exhibited direct interaction with cells in the culture, resulting in secretion of chemokines that interfered with the interaction of HIV-1 virions with CCR5, the coreceptor for HIV-1 on the susceptible cells, leading to inhibition of infection by HIV-1. Phosphoinositides that are recognized by WR321 do not exist on the external surface of cells, but are concentrated on the inner surface (cytoplasmic leaflet) of the plasma membrane. Murine anti-phosphoinositide mAbs similar to WR321 have previously been directly microinjected into a variety of cultured cells, resulting in important changes in the functions of the cells. The present results suggest that binding of a mAb to phosphoinositides, resulting in secretion of β-chemokines into the culture medium and neutralization of infection by CCR5-tropic HIV-1 of nearby susceptible cells, occurred by uptake and binding of the mAb at an intracellular location in the cultured cells that then led to secretion of HIV-1-inhibitory β-chemokines.     

Enhanced proliferation and migration of vascular smooth muscle cells in response to vascular injury under hyperglycemic conditions is controlled by β3 integrin signaling

Atheroma formation and restenosis following percutaneous vascular intervention involve the growth and migration of vascular smooth muscle cells (SMCs) into neointimal lesions, in part due to changes in the extracellular matrix. While some clinical studies have suggested that, in comparison to non-diabetics, β3 integrin inhibition in diabetic patients confers protection from restenosis, little is known regarding the role of β3 integrin inhibition on SMC responses in this context. To understand the molecular mechanisms underlying integrin-mediated regulation of SMC function in diabetes, we examined SMC responses in diabetic mice deficient in integrin β3 and observed that the integrin was required for enhanced proliferation, migration and extracellular regulated kinase (ERK) activation. Hyperglycemia-enhanced membrane recruitment and catalytic activity of PKCβ in an integrin β3-dependent manner. Hyperglycemia also promoted SMC filopodia formation and cell migration, both of which required αVβ3, PKCβ, and ERK activity. Furthermore, the integrin–kinase association was regulated by the αVβ3 integrin ligand thrombospondin and the integrin modulator Rap1 under conditions of hyperglycemia. These results suggest that there are differences in SMC responses to vascular injury depending on the presence or absence of hyperglycemia and that SMC response under hyperglycemic conditions is largely mediated through β3 integrin signaling.     

Norepinephrine promotes the β1-integrin-mediated adhesion of MDA-MB-231 cells to vascular endothelium by the induction of a GROα release

The migratory activity of tumor cells and their ability to extravasate from the blood stream through the vascular endothelium are important steps within the metastasis cascade. We have shown previously that norepinephrine is a potent inducer of the migration of MDA-MB-468 human breast carcinoma cells and therefore investigated herein, whether the interaction of these cells as well as MDA-MB-231 and MDA-MB-435S human breast carcinoma cells with the vascular endothelium is affected by this neurotransmitter as well. By means of a flow-through assay under physiologic flow conditions, we show that norepinephrine induces an increase of the adhesion of the MDA-MB-231 cells, but not of MDA-MB-468 and MDA-MB-435S cells to human pulmonary microvascular endothelial cells (HMVEC). The adhesion of MDA-MB-231 cells was based on a norepinephrine-mediated release of GROα from HMVECs. GROα caused a β1-integrin-mediated increase of the adhesion of MDA-MB-231 cells. Most interestingly, this effect of norepinephrine, similar to the aforementioned induction of migration in MDA-MB-468 cells, was mediated by β-adrenergic receptors and therefore abrogated by β-blockers. In conclusion, norepinephrine has cell line-specific effects with regard to certain steps of the metastasis cascade, which are conjointly inhibited by clinically established β-blockers. Therefore, these results may deliver a molecular explanation for our recently published retrospective data analysis of patients with breast cancer which shows that β-blockers significantly reduce the development of metastases.     

Fibrillar collagen type I stimulation of apolipoprotein B secretion in Caco-2 cells is mediated by β1 integrin

Caco-2 cells spontaneously differentiate into enterocyte-like cells and secrete apolipoprotein B (apoB) lipoproteins. We evaluated the effect of different extracellular matrix proteins on lipoprotein secretion by these cells. Caco-2 cells grown on human amnion connective tissue (HACT) secreted twice as much apoB as control cells on Transwells, but secreted similar amounts of apoA1. Cells cultured on fibrillar collagen type I secreted increased amounts of apoB similar to the cells cultured on HACT, but cells cultured on non-fibrillar collagen type I, type IV collagen or laminin-1 did not. The increased secretion was nullified by a function inhibiting anti-integrin β1 monoclonal antibody. Therefore, interactions between type I collagen and β1 integrins augment apoB secretion by Caco-2 cells. Cells on HACT formed a more uniform columnar epithelium with lipid droplets polarized to the basolateral membrane. We also studied the effect of extracellular matrix proteins on transepithelial resistance (TER) of differentiated Caco-2 cells. TER in cells cultured on HACT was similar to that on Transwells, but cells on laminin-1 and collagen IV exhibited higher TER. Thus, various extracellular matrix proteins regulate apoB secretion and TER differently. This new observation that extracellular matrix proteins can enhance apoB secretion in Caco-2 cells could be useful to explore the modulation of lipid transport by these proteins.     

Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.     

Sorting nexin 17 prevents lysosomal degradation of β1 integrins by binding to the β1-integrin tail

Integrin functions are controlled by regulating their affinity for ligand, and by the efficient recycling of intact integrins through endosomes. Here we demonstrate that the Kindlin-binding site in the β1-integrin cytoplasmic domain serves as a molecular switch enabling the sequential binding of two FERM-domain-containing proteins in different cellular compartments. When β1 integrins are at the plasma membrane, Kindlins control ligand-binding affinity. However, when they are internalized, Kindlins dissociate from integrins and sorting nexin 17 (SNX17) is recruited to free β1-integrin tails in early endosomes to prevent β1-integrin degradation, leading to their recycling back to the cell surface. Our results identify SNX17 as a β1-integrin-tail-binding protein that interacts with the free Kindlin-binding site in endosomes to stabilize β1 integrins, resulting in their recycling to the cell surface where they can be reused.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

mTORC1 signaling under hypoxic conditions is controlled by ATM-dependent phosphorylation of HIF-1α

The mTOR complex-1 (mTORC1) coordinates cell growth and metabolism, acting as a restriction point under stress conditions such as low oxygen tension (hypoxia). Hypoxia suppresses mTORC1 signaling. However, the signals by which hypoxia suppresses mTORC1 are only partially understood, and a direct link between hypoxia-driven physiological stress and the regulation of mTORC1 signaling is unknown. Here we show that hypoxia results in ataxia telangiectasia mutated (ATM)-dependent phosphorylation of hypoxia-inducible factor 1-alpha (HIF-1α) on serine(696) and mediates downregulation of mTORC1 signaling. Deregulation of these pathways in pediatric solid tumor xenografts suggests a link between mTORC1 dysregulation and solid tumor development and points to an important role for hypoxic regulation of mTORC1 activity in tumor development.     

Modulation of membrane properties of lung cancer cells by azurin enhances the sensitivity to EGFR-targeted therapy and decreased β1 integrin-mediated adhesion

In lung cancer, the Epidermal Growth Factor Receptor (EGFR) is one of the main targets for clinical management of this disease. The effectiveness of therapies toward this receptor has already been linked to the expression of integrin receptor subunit β₁ in NSCLC A549 cells. In this work we demonstrate that azurin, an anticancer therapeutic protein originated from bacterial cells, controls the levels of integrin β₁ and its appropriate membrane localization, impairing the intracellular signaling cascades downstream these receptors and the invasiveness of cells. We show evidences that azurin when combined with gefitinib and erlotinib, tyrosine kinase inhibitors which targets specifically the EGFR, enhances the sensitivity of these lung cancer cells to these molecules. The broad effect of azurin at the cell surface level was examined by Atomic Force Microscopy. The Young 's module (E) shows that the stiffness of A549 lung cancer cells decreased with exposure to azurin and also gefitinib, suggesting that the alterations in the membrane properties may be the basis of the broad anticancer activity of this protein. Overall, these results show that azurin may be relevant as an adjuvant to improve the effects of other anticancer agents already in clinical use, to which patients often develop resistance hampering its full therapeutic response     

Expression and secretion of β-glucuronidase and Pertussis toxin S1 by Streptomyces lividans

 Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coliβ-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the β-glucuronidase gene was fused with the leader sequence produced up to 30 mg β-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/l. The disappearence of the B. pertussis toxin S1 and β-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans. Received: 19 July 1995/Received revision: 3 November 1995/Accepted: 20 November 1995     

Resistance of Soybean Pectin–Protein Conjugate Pre-Adsorbed to the Air–Water Interface to Displacement by the Competitive Adsorption of Surfactant

Food Biophysics - The naturally occurring soybean pectin–protein conjugate pre-adsorbed to the air–water interface was shown to be displaced competitively from the interface when a...     

PTH stimulates the proliferation of TE-85 human osteosarcoma cells by a mechanism not involving either increased cAMP or increased secretion of IGF-I,IGF-II or TGFβ

Injections of parathyroid hormone (PTH) result in increased bone formation in several species. Work in our laboratory and others has shown a stimulation of bone cell proliferation and growth factor production by PTH. Our purpose was to study the effects of PTH on a human bone cell line using TE-85 human osteosarcoma cells as a model. After 24 h treatment, PTH caused an increase in cell proliferation as measured by cell counts and [³H]-thymidine incorporation. Proliferation was not inhibited by an anti-transforming growth factor beta (TGFβ) antibody which could abolish stimulation by exogenous TGFβ. PTH did not stimulate cAMP production, alkaline phosphatase activity or production of insulin-like growth factors I or II (IGF-I or IGF-II) in TE-85 cells. Although basal TE-85 proliferation was slowed by incubation with the calcium channel blocking agent verapamil, PTH still caused an increase in growth rate. We conclude that PTH directly stimulates TE-85 proliferation via a mechanism not involving increased adenylate cyclase activity or increased secretion of IGF-I, IGF-II or TGFβ and may stimulate bone formation in vivo by activating some other mitogenic signal to increase bone cell proliferation.