Dysfunctional telomeres limit cellular proliferative capacity by activating the p53-p21– and p16INK4a-Rb–dependent DNA damage responses (DDRs). The p16INK4a tumor suppressor accumulates in aging tissues, is a biomarker for cellular senescence, and limits stem cell function in vivo. While the activation of a p53-dependent DDR by dysfunctional telomeres has been well documented in human cells and mouse models, the role for p16INK4a in response to telomere dysfunction remains unclear. Here, we generated protection of telomeres 1b
Yang Wang, Norman Sharpless, Sandy Chang
Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug–induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with
Lindsey A. May, Inga I. Kramarenko, Carlene S. Brandon, Christina Voelkel-Johnson, Soumen Roy, Kristy Truong, Shimon P. Francis, Elyssa L. Monzack, Fu-Shing Lee, Lisa L. Cunningham
Breast cancers commonly become resistant to EGFR–tyrosine kinase inhibitors (EGFR-TKIs); however, the mechanisms of this resistance remain largely unknown. We hypothesized that resistance may originate, at least in part, from molecular alterations that activate signaling downstream of EGFR. Using a screen to measure reversion of malignant cells into phenotypically nonmalignant cells in 3D gels, we identified FAM83A as a candidate cancer-associated gene capable of conferring resistance to EGFR-TKIs. FAM83A overexpression in cancer cells increased proliferation and invasion and imparted EGFR-TKI resistance both in cultured cells and in animals. Tumor cells that survived EGFR-TKI treatment in vivo had upregulated FAM83A levels. Additionally, FAM83A overexpression dramatically increased the number and size of transformed foci in cultured cells and anchorage-independent growth in soft agar. Conversely, FAM83A depletion in cancer cells caused reversion of the malignant phenotype, delayed tumor growth in mice, and rendered cells more sensitive to EGFR-TKI. Analyses of published clinical data revealed a correlation between high FAM83A expression and breast cancer patients’ poor prognosis. We found that FAM83A interacted with and caused phosphorylation of c-RAF and PI3K p85, upstream of MAPK and downstream of EGFR. These data provide an additional mechanism by which tumor cells can become EGFR-TKI resistant.
Sun-Young Lee, Roland Meier, Saori Furuta, Marc E. Lenburg, Paraic A. Kenny, Ren Xu, Mina J. Bissell
Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical for the inhibitory actions of Cav-1 toward eNOS. Herein, we show that F92A–Cav-1 and a mutant cell–permeable scaffolding domain peptide called Cavnoxin can increase basal NO release in eNOS-expressing cells. Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed with eNOS- or Cav-1–deficient mice showed that the vasodilatory effect of Cavnoxin is abolished in the absence of these gene products, which indicates a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that noninhibitory F92A–Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with the other actions of endogenous Cav-1. They also suggest a therapeutic application for regulating the eNOS/Cav-1 interaction in diseases characterized by decreased NO release.
Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa
GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P1) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.
Myat Lin Oo, Sung-Hee Chang, Shobha Thangada, Ming-Tao Wu, Karim Rezaul, Victoria Blaho, Sun-Il Hwang, David K. Han, Timothy Hla
Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft–associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endosomal sorting of the toxin-GM1 complex.
David E. Saslowsky, Jin Ah Cho, Himani Chinnapen, Ramiro H. Massol, Daniel J.-F. Chinnapen, Jessica S. Wagner, Heidi E. De Luca, Wendy Kam, Barry H. Paw, Wayne I. Lencer
Fibroblasts are ubiquitous cells that demonstrate remarkable diversity. However, their origin and pathways of differentiation remain poorly defined. Here, we show that connective tissue growth factor (CTGF; also known as CCN2) is sufficient to induce human bone marrow mesenchymal stem/stromal cells (MSCs) to differentiate into fibroblasts. CTGF-stimulated MSCs lost their surface mesenchymal epitopes, expressed broad fibroblastic hallmarks, and increasingly synthesized collagen type I and tenacin-C. After fibroblastic commitment, the ability of MSCs to differentiate into nonfibroblastic lineages — including osteoblasts, chondrocytes, and adipocytes — was diminished. To address inherent heterogeneity in MSC culture, we established 18 single MSC–derived clones by limiting dilution. CTGF-treated MSCs were α-SMA–, differentiating into α-SMA+ myofibroblasts only when stimulated subsequently with TGF-β1, suggestive of stepwise processes of fibroblast commitment, fibrogenesis, and pathological fibrosis. In rats, in vivo microencapsulated delivery of CTGF prompted postnatal connective tissue to undergo fibrogenesis rather than ectopic mineralization. The knowledge that fibroblasts have a mesenchymal origin may enrich our understanding of organ fibrosis, cancer stroma, ectopic mineralization, scarring, and regeneration.
Chang H. Lee, Bhranti Shah, Eduardo K. Moioli, Jeremy J. Mao
Hereditary spastic paraplegias (HSPs; SPG1–45) are inherited neurological disorders characterized by lower extremity spastic weakness. More than half of HSP cases result from autosomal dominant mutations in atlastin-1 (also known as SPG3A), receptor expression enhancing protein 1 (REEP1; SPG31), or spastin (SPG4). The atlastin-1 GTPase interacts with spastin, a microtubule-severing ATPase, as well as with the DP1/Yop1p and reticulon families of ER-shaping proteins, and SPG3A caused by atlastin-1 mutations has been linked pathogenically to abnormal ER morphology. Here we investigated SPG31 by analyzing the distribution, interactions, and functions of REEP1. We determined that REEP1 is structurally related to the DP1/Yop1p family of ER-shaping proteins and localizes to the ER in cultured rat cerebral cortical neurons, where it colocalizes with spastin and atlastin-1. Upon overexpression in COS7 cells, REEP1 formed protein complexes with atlastin-1 and spastin within the tubular ER, and these interactions required hydrophobic hairpin domains in each of these proteins. REEP proteins were required for ER network formation in vitro, and REEP1 also bound microtubules and promoted ER alignment along the microtubule cytoskeleton in COS7 cells. A SPG31 mutant REEP1 lacking the C-terminal cytoplasmic region did not interact with microtubules and disrupted the ER network. These data indicate that the HSP proteins atlastin-1, spastin, and REEP1 interact within the tubular ER membrane in corticospinal neurons to coordinate ER shaping and microtubule dynamics. Thus, defects in tubular ER shaping and network interactions with the microtubule cytoskeleton seem to be the predominant pathogenic mechanism of HSP.
Seong H. Park, Peng-Peng Zhu, Rell L. Parker, Craig Blackstone
The RNA-binding protein HuR (also known as ELAV1) binds to the 3′-untranslated region of mRNAs and regulates transcript stability and translation. However, the in vivo functions of HuR are not well understood. Here, we report that murine HuR is essential for life; postnatal global deletion of Elavl1 induced atrophy of hematopoietic organs, extensive loss of intestinal villi, obstructive enterocolitis, and lethality within 10 days. Upon Elavl1 deletion, progenitor cells in the BM, thymus, and intestine underwent apoptosis, whereas quiescent stem cells and differentiated cells were unaffected. The survival defect of hematopoietic progenitor cells was cell intrinsic, as transplant of Elavl1–/– BM led to compromised hematopoietic reconstitution but did not cause lethality. Expression of p53 and its downstream effectors critical for cell death were induced in progenitor cells as HuR levels declined. In mouse embryonic fibroblasts, HuR bound to and stabilized the mRNA for Mdm2, a critical negative regulator of p53. Furthermore, cell survival was restored by expression of Mdm2 in Elavl1–/– cells, suggesting that HuR keeps p53 levels in check in progenitor cells and thereby promotes cell survival. This regulation of cell stress response by HuR in progenitor cells, which we believe to be novel, could potentially be exploited in cytotoxic anticancer therapies as well as stem cell transplant therapy.
Mallika Ghosh, Hector Leonardo Aguila, Jason Michaud, Youxi Ai, Ming-Tao Wu, Annabrita Hemmes, Ari Ristimaki, Caiying Guo, Henry Furneaux, Timothy Hla
ER stress–induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress–mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress–induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and JNK. Remarkably, CaMKIIγ was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress–induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIγ-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress–induced apoptosis.
Jenelle M. Timmins, Lale Ozcan, Tracie A. Seimon, Gang Li, Cristina Malagelada, Johannes Backs, Thea Backs, Rhonda Bassel-Duby, Eric N. Olson, Mark E. Anderson, Ira Tabas
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