We previously reported that exposure of human airway epithelial cells to oxidative stress increased ceramide generation via specific activation of neutral sphingomyelinase2 (nSMase2). Here we show that nSMase2 is a phosphoprotein exclusively phosphorylated at serine residues. The level of nSMase2 phosphorylation can be modulated by treatment with anisomycin or phorbol 12-myristate 13-acetate (PMA/12-O-tetradecanoylphorbol-13-acetate), suggesting that p38 mitogen-activated protein kinase (MAPK) and protein kinases Cs are upstream of nSMase2 phosphorylation. Oxidative stress enhances both the activity and phosphorylation of nSMase2. Strikingly, we show here that nSMase2 is bound directly by the phosphatase calcineurin (CaN), which acts as an on/off switch for nSMase2 phosphorylation in the presence or absence of oxidative stress. Specifically, CaN is being inhibited/degraded and therefore does not bind nSMase2 under oxidative stress, and a mutant nSMase2 that lacks the CaN binding site exhibits constitutively elevated phosphorylation and increased activity relative to wild type nSMase2. Importantly, the phosphorylation and activity of the mutant no longer responds to oxidative stress, confirming that CaN is the critical link that allows oxidative stress to modulate nSMase2 phosphorylation and function.We have shown that ceramide generation coordinates stress responses and is elevated in HAE cells in response to reactive oxygen species (1-8).Ceramide is synthesized through either a de novo pathway involving serine palmitoyl-CoA transferase and ceramide synthase, or from breakdown of membrane sphingomyelin (N-acylsphingosine-1-phosphocholine) (Fig. 1A), a phospholipid preferentially concentrated in the plasma membrane of mammalian cells (9). Sphingomyelin catabolism occurs via the action of sphingomyelinases (SMases), 3 which are sphingomyelin-specific forms of phospholipase C that hydrolyze the phosphodiester bond of sphingomyelin, yielding ceramide and phosphorylcholine. Ceramide then serves as a second messenger, leading to apoptotic DNA degradation.We suggested that reactive oxidants up-regulate ceramide generation and cause elevated apoptosis in human airway epithelial (HAE) cells, thereby leading to lung injury pathologies. However, the mechanisms and target molecules of reactive oxidants affecting the HAE cells are not fully understood. Therefore, we proposed that increased oxidative stress and elevated ceramide generation are coupled at the molecular level by an unknown SMase that generates ceramide by hydrolysis of sphingomyelin (Fig. 1B). To proceed from the cellular to the molecular level, we searched for the specific SMase that is modulated by reactive oxygen species in lung epithelial cells, which led to our isolation of the novel nSMase2 from monkey lung tissue and HAE cells (1). This nSMase2 was previously found in the brain (10).We then demonstrated that nSMase2 is the only member of the sphingomyelin phosphodiesterases family that is up-regulated and responsible for ceramide generation in HAE cells ...
Aggressive behavior among females is observed in many species, but the mechanisms of this behavior have historically been understudied. In many species of rodents, winter-like short day photoperiods induce increased aggression levels compared to summer-like long day photoperiods. Recent reports in hamsters show that short days also increase aggression in females. We examined the effects of photoperiod on aggression in female California mice, and for the first time compare brain activity of aggression-tested female rodents under different photoperiods. We observed that female California mice were more aggressive when housed in short days versus long days. Intriguingly, we also observed that under long days female attack latency decreases with repeated testing in resident intruder tests. These data suggest that winner effects that have been described in males may also occur in females. We also used the expression of phosphorylated extracellular signal-regulated kinases (pERK) in the brain to estimate brain activity during aggression tests. pERK can alter neuronal activity in the short term and in the long term can act as a transcription factor. Using immunoblot analyses we observed that aggression-induced pERK expression in the female bed nucleus of the stria terminalis (BNST) and medial amygdala occurs under both long and short days. Thus, the mechanisms controlling increased aggression under short days are still unclear and additional study is needed.
The ErbB3 receptor tyrosine kinase contributes to a variety of developmental processes, and its overexpression and aberrant activation promote tumor progression and therapeutic resistance. Accumulating evidence suggests that tumor overexpression may be mediated by the loss of posttranscriptional negative regulatory mechanisms, such as protein degradation, that normally keep receptor levels in check. Our previous studies indicate that the RING finger E3 ubiquitin ligase Nrdp1, a protein lost in breast and other tumor types, suppresses ErbB3 levels by mediating ligand-independent receptor ubiquitination and degradation. Here we demonstrate that Nrdp1 preferentially associates with the nascent form of ErbB3 to accelerate its degradation, and we show that the two proteins colocalize at the endoplasmic reticulum (ER). Blocking the exit of ErbB3 from the ER does not affect the ability of Nrdp1 to mediate receptor ubiquitination or degradation, while functional disruption of the conserved ER-associated degradation (ERAD) pathway ATPase VCP/p97 leads to the Nrdp1-dependent accumulation of ubiquitinated ErbB3 but blocks receptor degradation. Further evidence indicates that the ErbB3 targeted by Nrdp1 for degradation is properly folded and fully functional. Collectively, these observations point to a novel mechanism of receptor tyrosine kinase quantity control wherein steadystate levels of signaling-competent receptor are dictated by an ER-localized degradation pathway.
We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFRneutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most upregulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.colorectal cancer | versican | HPGD | 3D culture | cetuximab resistance T raditionally, epithelial cells have been cultured on plastic as a flat monolayer, precluding formation of their characteristic apico-basolateral structural organization. Pioneering work by Mina Bissell and Joan Brugge has shown that select breast epithelial cell lines can be grown in 3D in Matrigel as polarizing cysts with intact apico-basolateral polarity (1-3). These 3D cultures have been used to study oncogene-induced transformation and are shown to better predict in vivo behavior than 2D cultures (4, 5). Similar work in colonic epithelial cells has lagged behind; a notable exception is the work of Alan Hall and colleagues with Caco-2 cells that form uniform polarizing cysts in 3D Matrigel (6).We sought to identify human colorectal cancer (CRC) lines that exhibit apico-basolateral polarity in a better-defined 3D environment than Matrigel, which is a complex, incompletely defined extracellular matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (7). We previously observed that a human CRC line, HCA-7, cultured in type-1 collagen, gave rise to colonies consisting of unilamellar cysts with intact apico-basolateral polarity or less frequent colonies composed of irregular solid masses of cells (8). We derived CC and SC lines from cystic and spiky colonies, respectively. When injected sub...
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