Zhenyi Ma

Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local ras activation

ABSTRACT The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H2O2 sensor targeted to different subcellular compartments, we demonstrate specific production of H2O2 by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H2O2 production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.

Subcellular targeting of oxidants during endothelial cell migration

Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47phox and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

HIV-1 Tat Activates Dual Nox Pathways Leading to Independent Activation of ERK and JNK MAP Kinases

Human immunodeficiency virus, type 1 Tat is known to exert pleiotropic effects on the vascular endothelium through mitogen-activated protein (MAP) kinases, although the signaling pathways leading to MAP kinase activation are incompletely understood. We focused on proximal pathways potentially governing downstream MAP kinase activity by Tat. Within 2 min, Tat activated both Ras and Rho GTPases in endothelial cells, leading to ERK phosphorylation by 10 min. Notably, Rac1 was necessary for downstream activation of RhoA and both Rac1 and RhoA acted upstream of the Ras/ERK cassette. Antioxidants and the oxidase inhibitor diphenylene iodonium blocked ERK phosphorylation, but specific interference with the canonical Nox2 oxidase had no effect on ERK. Instead, knock down of the novel oxidase Nox4 completely suppressed Tat-dependent Ras and ERK activation downstream of Rac1 and RhoA. Conversely, interference with Rac1, PAK1, and Nox2 blocked JNK phosphorylation, whereas RhoA(N19) and Nox4 knock down did not. Further, knock down of Nox2, but not Nox4, blocked Tat-induced cytoskeletal rearrangement, whereas knock down of Nox4, but not Nox2, blocked Tat-dependent proliferation. Rac1, therefore, bifurcates Tat signaling, leading to concurrent but separate Nox4-dependent Ras/ERK activation, and Nox2-dependent JNK activation. Tat signaling, therefore, provides an example of Nox-specific differential control of MAP kinase pathways.

p66Shc mediates anoikis through RhoA

Detachment of parenchymal cells from a solid matrix switches contextual cues from survival to death during anoikis. Marked shape changes accompany detachment and are thought to trigger cell death, although a working model to explain the coordination of attachment sensation, shape change, and cell fate is elusive. The constitutive form of the adapter Shc, p52Shc, confers survival properties, whereas the longer p66Shc signals death through association with cytochrome c. We find that cells that lack p66Shc display poorly formed focal adhesions and escape anoikis. However, reexpression of p66Shc restores anoikis through a mechanism requiring focal adhesion targeting and RhoA activation but not an intact cytochrome c–binding motif. This pathway stimulates the formation of focal adhesions and stress fibers in attached cells and tension-dependent cell death upon detachment. p66Shc may thus report attachment status to the cell by imposing a tension test across candidate anchorage points, with load failure indicating detachment.

p66Shc restrains Ras hyperactivation and suppresses metastatic behavior

Normal tissue cells survive and proliferate only while anchored to solid substrate. Conversely, transformed cells both survive and proliferate following detachment, having lost attachment context through unclear mechanisms. p66Shc is a focal adhesion-associated protein that reports cell attachment through a RhoA-dependent mechanosensory test. We find that human small cell lung cancer (SCLC) cells and mouse Lewis lung carcinoma (LLC), which display aggressive metastatic behavior, lack both p66Shc and retinoblastoma (pRB) and bypass anoikis. Re-expression of p66Shc in these cells restores anoikis and provides striking protection from metastasis by LLC cells in vivo. Notably, knockdown of p66Shc in normal epithelial cells leads to unrestrained Ras activation, preventing anoikis through downstream suppression of RhoA but blocking proliferation in a pRB-dependent manner, thus mimicking oncogenic Ras. Conversely, LLC and SCLC cells display constitutive Ras activation necessary to bypass anoikis, which is reversed by re-expression of p66Shc. p66Shc therefore coordinates Ras-dependent control of proliferation and anchorage sensation, which can be defeated in the evolution of highly metastatic tumors by combined loss of both p66Shc and pRB.

Feedback loop between p66Shc and Nrf2 promotes lung cancer progression

p66(Shc), one of the SHC1 gene encoding proteins, promotes cell death and reports cell anchorage status, mediating anoikis in vitro and functioning as a metastasis suppressor in vivo. However, very little is known about p66(Shc) gene regulation in cancer cells. Here, we show that methylation of a specific CpG site in the early post-transcriptional region correlates with p66(Shc) repression in clinical human lung cancer samples and cancer cell lines. We also find that the stress related transcription factor Nrf2 associates with p66(Shc) gene promoter in the methylated region, and promotes p66(Shc) transcription. However, p66(Shc) induction by Nrf2 requires demethylation of the Nrf2 binding site in p66(Shc) promoter. Knock-down of p66(Shc) leads to a positive feedback upregulation of Nrf2 expression and accordingly, Nrf2 is found to be highly expressed in tumors with low p66(Shc) expression. Further, Nrf2 expression level positively correlates with tumor grade of patients. Thus, we propose that epigenetic repression of p66(Shc) in cancer cells might be a key factor leading to Nrf2 upregulation, increased cell survival, and tumor progression.

Downregulated adaptor protein p66Shc mitigates autophagy process by low nutrient and enhances apoptotic resistance in human lung adenocarcinoma A549 cells

Macroautophagy or autophagy is a lysosome‐dependent process in which enzymatic degradation and recycling of cytosolic components occur in stressful contexts. The mechanisms underlying the signaling from starvation to the regulation of autophagy are not fully understood. We previously showed that the Src family member p66Shc (focal adhesion‐associated 66 kDa isoform of the Src homology and collagen) promotes anoikis and suppresses tumor metastasis via k‐Ras‐dependent control of proliferation and survival. However, the role of p66Shc in low‐nutrient‐induced autophagy‐related pathways remains elusive. In this work, human lung adenocarcinoma A549 cells were used to further investigate the biological effects of p66Shc on autophagy and apoptotic resistance. Here, we show that deficiency of p66Shc mitigates the low‐nutrient‐induced autophagy process in the levels of microtubule‐associated protein 1A light chain protein 3B (LC3B) conversion, in the number of autophagic vacuoles and in p62/sequestosome 1 protein degradation. However, autophagy‐related protein Beclin 1 was not significantly changed during low‐nutrient treatment. Furthermore, we found that prolonged phosphorylation of extracellular signaling‐regulated kinase (Erk)1/2, but not phosphorylation of Akt is significantly sustained when p66Shc expression is inhibited by shRNA. In addition, cleavage of caspase 7 and poly(ADP‐ribose) polymerase, but not caspase 6 and 9 are retarded with this effect compared to the shRNA control cells. Together, these findings suggest the possibility that p66Shc plays a pivotal role in coordinately regulating autophagy process and apoptotic resistance in A549 cells under nutrient‐limited conditions.

Divergent Roles of RelA and c-Rel in Establishing Chromosomal Loops upon Activation of the Igκ Gene

Precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening DNA, but how trans-acting regulatory proteins work to establish and maintain DNA loops during gene activation remains largely unexplored. LPS-induced transcription of the mouse Igκ gene in B lymphocytes utilizes three distal enhancers and requires the transcription factor NF-κB, whose family members include RelA and c-Rel. Using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that LPS-induced Igκ gene activation creates chromosomal loops by bridging together all three pairwise interactions between the distal enhancers and RNA polymerase II, the apparent molecular tie for the bases of these loops. RelA and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis, or c-Rel. We have thus identified both essential and nonessential events that establish higher order chromatin reorganization during Igκ gene activation.

Integration of Autophagy and Anoikis Resistance in Solid Tumors

Macroautophagy or autophagy is a lysosome‐dependent process in which enzymatic degradation and recycling of cytosolic components occurred due to stressful conditions. This cellular arrangement imparts anoikis resistance in solid tumors. Anoikis, a special form of apoptosis occurring when cells detach from the extracellular matrix, is a critical mechanism in maintaining tissue homeostasis and development. Anoikis resistance facilitates tumorigenesis and metastasis. However, the complexity of the role of autophagy in tumor is underscored by evidence that autophagy can function as both a pro‐survival or pro‐death depending on the context and the stimuli, which are likely exploitable for tumor therapy. This review focuses on recent progress in understanding anoikis resistance and autophagy signaling, paying particular attention to its relevance in solid tumor metastasis. Anat Rec, 296:1501–1508, 2013.

Mechanotransduction and anoikis: death and the homeless cell.

Developed organs display strict spatial organization of differentiated cells which is required for proper organ function. One important device that prevents tissue disorganization is the death of cells that lose anchorage to their native matrix, a signal that indicates potential loss of proper tissue context. Termed anoikis (Greek for Homelessness), this form of cell death is a specialized form of apoptosis. Interestingly, at certain stages of development and tissue repair, cells are required to migrate in an unanchored state, suggesting that anoikis must be strictly regulated at some level. Likewise, cellular transformation is often accompanied by an inappropriate loss of anoikis and subsequent acquisition of a metastatic phenotype. Despite its importance, the molecular pathways involved in the regulation of anoikis and the proximal signals reporting loss of anchorage are poorly understood. Recent studies suggest that attachment may be reported by a mechanosensory testing of the cells physical environment.