Analysis of inhibitor of apoptosis protein family expression during mammary gland development

<p>Abstract</p> <p>Background</p> <p>Inhibitors-of-Apoptosis-Proteins (IAPs) are an evolutionarily conserved family of proteins capable of regulating several facets of apoptosis. IAPs are frequently dysregulated in cancer, but their role in the regulation of apoptosis during developmental processes is not fully understood. Here we examined the expression of IAPs during the post-natal development of the mouse mammary gland, which is a tissue that exhibits a profound induction of apoptosis during involution.</p> <p>Results</p> <p>Six out of eight mammalian IAP family members are expressed in the mammary gland. Notably, quantitative PCR and immunoblotting revealed that XIAP, c-IAP1 and c-IAP2 are down-regulated in pregnancy and lactation, and prior to the onset of involution. In cultured mammary epithelial cells (MECs), XIAP levels decreased in response to inhibition of growth factor signalling. Maintaining XIAP levels in MECs by expressing exogenous XIAP protected them from all apoptotic stimuli tested.</p> <p>Conclusions</p> <p>These data suggest that the developmental regulation of IAP expression <it>in vivo </it>contributes to naturally occurring programmes of cell death.</p

Sodium channel gene family: epilepsy mutations, gene interactions and modifier effects

The human sodium channel family includes seven neuronal channels that are essential for the initiation and propagation of action potentials in the CNS and PNS. In view of their critical role in neuronal firing and their strong sequence conservation during evolution, it is not surprising that mutations in the sodium channel genes are responsible for a growing spectrum of channelopathies. Nearly 700 mutations of the SCN1A gene have been identified in patients with Dravet's syndrome (severe myoclonic epilepsy of infancy), making this the most commonly mutated gene in human epilepsy. A small number of mutations have been found in SCN2A , SCN3A and SCN9A , and studies in the mouse suggest that SCN8A may also contribute to seizure disorders. Interactions between genetic variants of SCN2A and KCNQ2 in the mouse and variants of SCN1A and SCN9A in patients provide models of potential genetic modifier effects in the more common human polygenic epilepsies. New methods for generating induced pluripotent stem cells and neurons from patients will facilitate functional analysis of amino acid substitutions in channel proteins. Whole genome sequencing and exome sequencing in patients with epilepsy will soon make it possible to detect multiple variants and their interactions in the genomes of patients with seizure disorders.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79388/1/jphysiol.2010.188482.pd

The RhoA-Rok-myosin II pathway is involved in extracellular matrix-mediated regulation of prolactin signaling in mammary epithelial cells

In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell–BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell–matrix interactions

Angiogenic Functions of Voltage-gated Na+ Channels in Human Endothelial Cells: MODULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) SIGNALING*

Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and β-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCβ1, and VGSCβ3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca2+]i. We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis

Lung cancer:intragenic ERBB2 kinase mutations in tumours

The protein-kinase family is the most frequently mutated gene family found in human cancer and faulty kinase enzymes are being investigated as promising targets for the design of antitumour therapies. We have sequenced the gene encoding the transmembrane protein tyrosine kinase ERBB2 (also known as HER2 or Neu) from 120 primary lung tumours and identified 4% that have mutations within the kinase domain; in the adenocarcinoma subtype of lung cancer, 10% of cases had mutations. ERBB2 inhibitors, which have so far proved to be ineffective in treating lung cancer, should now be clinically re-evaluated in the specific subset of patients with lung cancer whose tumours carry ERBB2 mutations