Truncation mutants of the tight junction protein ZO-1 disrupt corneal epithelial cell morphology

The tight junction is the most apical intercellular junction of epithelial cells and regulates transepithelial permeability through the paracellular pathway. To examine possible functions for the tight junction-associated protein ZO-1, C-terminally truncated mutants and a deletion mutant of ZO-1 were epitope tagged and stably expressed in corneal epithelial cell lines. Only full-length ZO-1 and one N-terminal truncation mutant targeted to cell borders; other mutants showed variable cytoplasmic distributions. None of the mutants initially disrupted the localization of endogenous ZO-1. However, long-term stable expression of two of the N-terminal mutants resulted in a dramatic change in cell shape and patterns of gene expression. An elongated fibroblast-like shape replaced characteristic epithelial cobblestone morphology. In addition, vimentin and smooth muscle actin expression were up-regulated, although variable cytokeratin expression remained, suggesting a partial transformation to a mesenchymal cell type. Concomitant with the morphological change, the expression of the integral membrane tight junction protein occludin was significantly down-regulated. The localizations of endogenous ZO-1 and another family member, ZO-2, were disrupted. These findings suggest that ZO-1 may participate in regulation of cellular differentiation.     

Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells, alveolar macrophages, type II pneumocytes, and neutrophils.

Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that occur in many mammalian cell types. Because lipid bodies are more prominent in cells associated with inflammation and are repositories of arachidonyl-phospholipids, a role for lipid bodies in the oxidative metabolism of arachidonic acid to form eicosanoids has been suggested. To evaluate further whether lipid bodies, in addition to serving as non-membranous sources of substrate arachidonate, are involved in eicosanoid formation, we used cells isolated from human lung to investigate the intracellular localization of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase), the key initial, rate-limiting enzyme in the formation of prostaglandins and thromboxanes. Isolated lung cells containing a mixture of mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils from short-term cultures were fixed in suspension in a dilute aldehyde mixture, post-fixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in a graded series of alcohols, and embedded in Epon. A post-embedding immunogold procedure was used with a primary PGH synthase monoclonal antibody and 20-nm gold-conjugated secondary antibody to demonstrate enzyme locations. Specificity controls were also done. We found PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils. Specific secretory and lysosomal granules and plasma membranes did not express PGH synthase. Specificity controls, including omission of the primary antibody or substitution with an irrelevant antibody, were negative. Absorption of the specific PGH synthase antibody with purified solid-phase PGH synthase resulted in a marked reduction of label in lipid bodies of all four cell types. These findings establish the presence of PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils and, in concert with previous studies, suggest that these cytoplasmic lipid-rich organelles may be non-membrane sites of eicosanoid formation.     

Lipoxin recognition sites. Specific binding of labeled lipoxin A4 with human neutrophils.

Lipoxin A4 stimulates rapid lipid remodeling and a pertussis toxin-sensitive release of arachidonic acid in polymorphonuclear leukocytes (PMN) (Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Physiol. 143, 512-523) and has been shown to inhibit leukocyte responses in several systems. To examine the basis underlying these actions, we have prepared [11,12-3H]lipoxin A4 (LXA4) and characterized its interactions with human PMN. Time course studies (0-90 min) with intact PMN demonstrated cell association of 3H label which was specific and reversible. PMN bound [3H]LXA4 with a Kd of 0.5 +/- 0.3 nM, representing approximately 1,830 sites/PMN, and the Hill plot value of 1.9 suggests cooperative binding. [3H]LXA4 binding was stereoselective since neither leukotriene B4 (LTB4), lipoxin B4 (LXB4), (6S)-LXA4, 11-trans-LXA4, nor SKF 104353 competed for [3H]LXA4-specific binding while LTD4 and LTC4 partially competed. Subcellular fractionation revealed that specific binding with [3H]LXA4 was associated with membrane (42.1%)-, granule (34.5%)-, and nuclear (23.3%)-enriched fractions, a distribution distinct from that of [14,15-3H] LTB4 binding. [11,12-3H]LXA4-specific binding was modulated by guanosine analogs, suggesting the involvement of G proteins. A fluorescent LXA4 derivative (methyl-7-methoxycoumarin-LXA4) competed with [3H]LXA4 binding to intact PMN and showed specific and reversible binding as monitored by flow cytometric analysis. These results indicate that PMN possess specific recognition sites for LXA4 which may mediate its actions.     

PIK3R3, part of the regulatory domain of PI3K,is upregulated in sarcoma stem-like cells and promotes invasion,migration, and chemotherapy resistance

To identify drivers of sarcoma cancer stem-like cells (CSCs), we compared gene expression using RNA sequencing between HT1080 fibrosarcoma and SK-LMS-1 leiomyosarcoma spheroids (which are enriched for CSCs) compared with the parent populations. The most overexpressed survival signaling-related gene in spheroids was phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of PI3K, which functions in tumorigenesis and metastasis. In a human sarcoma microarray, PIK3R3 was also overexpressed by 4.1-fold compared with normal tissues. PIK3R3 inhibition using shRNA in the HT1080, SK-LMS-1, and DDLS8817 dedifferentiated liposarcoma in spheroids and in CD133+ cells (a CSC marker) reduced expression of CD133 and the stem cell factor Nanog and blocked spheroid formation by 61–71%. Mechanistic studies showed that in spheroid cells, PIK3R3 activated AKT and ERK signaling. Inhibition of PIK3R3, AKT, or ERK using shRNA or inhibitors decreased expression of Nanog, spheroid formation by 68–73%, and anchorage-independent growth by 76–91%. PIK3R3 or ERK1/2 inhibition similarly blocked sarcoma spheroid cell migration, invasion, secretion of MMP-2, xenograft invasion into adjacent normal tissue, and chemotherapy resistance. Together, these results show that signaling through the PIK3R3/ERK/Nanog axis promotes sarcoma CSC phenotypes such as migration, invasion, and chemotherapy resistance, and identify PIK3R3 as a potential therapeutic target in sarcoma.Subject terms: Cancer stem cells, Oncogenes, Sarcoma     

Lead-Induced Impairments in the Neural Processes Related to Working Memory Function

Background

It is well known that lead exposure induces neurotoxic effects, which can result in a variety of neurocognitive dysfunction. Especially, occupational lead exposures in adults are associated with decreases in cognitive performance including working memory. Despite recent advances in human neuroimaging techniques, the neural correlates of lead-exposed cognitive impairment remain unclear. Therefore, this study was aimed to compare the neural activations in relation to working memory function between the lead-exposed subjects and healthy controls.

Methodology/Principal Findings

Thirty-one lead-exposed subjects and 34 healthy subjects performed an n-back memory task during MRI scan. We performed fMRI using the 1-back and 2-back memory tasks differing in cognitive demand. Functional MRI data were analyzed using within- and between-group analysis. We found that the lead-exposed subjects showed poorer working memory performance during high memory loading task than the healthy subjects. In addition, between-group analyses revealed that the lead-exposed subjects showed reduced activation in the dorsolateral prefrontal cortex, ventrolateral prefrontal cortex, pre supplementary motor areas, and inferior parietal cortex.

Conclusions/Significance

Our findings suggest that functional abnormalities in the frontoparietal working memory network might contribute to impairments in maintenance and manipulation of working memory in the lead-exposed subjects.     

TORC1 is essential for NF1-associated malignancies

Inactivating mutations in NF1 underlie the prevalent familial cancer syndrome neurofibromatosis type 1 [1]. The NF1-encoded protein is a Ras GTPase-activating protein (RasGAP) [2]. Accordingly, Ras is aberrantly activated in NF1-deficient tumors; however, it is unknown which effector pathways critically function in tumor development. Here we provide in vivo evidence that TORC1/mTOR activity is essential for tumorigenesis. Specifically, we show that the mTOR inhibitor rapamycin potently suppresses the growth of aggressive NF1-associated malignancies in a genetically engineered murine model. However, in these tumors rapamycin does not function via mechanisms generally assumed to mediate tumor suppression, including inhibition of HIF-1alpha and indirect suppression of AKT, but does suppress the mTOR target Cyclin D1 [3]. These results demonstrate that mTOR inhibitors may be an effective targeted therapy for this commonly untreatable malignancy. Moreover, they indicate that mTOR inhibitors do not suppress all tumor types via the same mechanism, suggesting that current biomarkers that rely on HIF-1alpha suppression may not be informative for all cancers. Finally, our results reveal important differences between the effects of mTOR inhibition on the microvasculature in genetically engineered versus xenograft models and indicate that the former may be required for effective preclinical screening with this class of inhibitors.     

Cytoplasmic lipid bodies of neutrophils: formation induced by cis- unsaturated fatty acids and mediated by protein kinase C

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.