Myriam Gorospe

Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.

The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. In this study we investigated the factors controlling MAPK activation by HO and explored the impact of altering the pathways to kinase activation on cell survival following HO exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of HO treatment, whereupon rapid inactivation ensued. HO activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, HO increased the mRNA expression of MAPK-dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited HO stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by HO was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation mediates the initiation of signal transduction by HO. HO-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to HO toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to HO toxicity, while those expressing kinase-defective MEK were more sensitive, than cells expressing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by HO and suggest that ERK plays a critical role in cell survival following oxidant injury.

LincRNA-p21 Suppresses Target mRNA Translation

Mammalian long intergenic noncoding RNAs (lincRNAs) are best known for modulating transcription. Here we report a posttranscriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated in human cervical carcinoma HeLa cells, increasing its association with JUNB and CTNNB1 mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and β-catenin translation and increased the levels of these proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.

RNA-binding protein HuR enhances p53 translation in response to ultraviolet light irradiation

Exposure to short-wavelength UV light (UVC) strongly induces p53 expression. In human RKO colorectal carcinoma cells, this increase was not due to elevated p53 mRNA abundance, cytoplasmic export of p53 mRNA, or UVC-triggered stabilization of the p53 protein. Instead, p53 translation was potently enhanced after UVC irradiation. The 3′ UTR of p53 was found to be a target of the RNA-binding protein HuR in a UVC-dependent manner in vitro and in vivo. HuR-overexpressing RKO cells displayed elevated p53 levels, whereas cells expressing reduced HuR showed markedly diminished p53 abundance and p53 translation. Our results demonstrate a role for HuR in binding to the p53 mRNA and enhancing its translation.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation

Colorectal carcinoma RKO cells expressing reduced levels of the RNA‐binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G1, increasing in the cytoplasm during late G1, S and G2. The expression and half‐life of mRNAs encoding cyclins A and B1 similarly increased during S and G2, then declined, indicating that mRNA stabilization contributed to their cell cycle‐regulated expression. In gel‐shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3′‐untranslated regions of cyclins A and B1 formed RNA–protein complexes in a cell cycle‐dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti‐HuR antibodies supershifted such RNA–protein complexes. Importantly, the expression and half‐life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle‐dependent stabilization of mRNAs encoding cyclins A and B1.

Long Noncoding RNA MALAT1 Controls Cell Cycle Progression by Regulating the Expression of Oncogenic Transcription Factor B-MYB

The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle–regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation.

p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells.

The tumor suppressive effect of p53 is believed to be rooted in its two primary functions: the implementation of cellular growth arrest and the execution of apoptotic cell death. While p53-regulated expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 appears to be central for the implementation of G1 arrest, the participation of p21Waf1/Cip1 in p53-triggered cell death remains controversial. In the present study, overexpression of p53 in human melanoma SK-MEL-110 cells through use of an adenoviral expression vector (AdCMV.p53) was found to result in apoptosis, while similar infection of primary vascular smooth muscle cells (VSMC) instead resulted in a moderate inhibition of growth. Expression of p21Waf1/Cip1 was strongly elevated in VSMC, but showed little change in SK-MEL-110 cells, although expression of another p53-regulated gene (GADD45) was comparable in both AdCMV.p53-infected cell types. Evidence that p21Waf1/Cip1 expression may be required for surviving p53-induced cell death was further supported by the finding that p53 overexpression was highly toxic for p21-deficient mouse embryonal fibroblasts (p21−/− MEFs). In both SK-MEL-110 and p21−/− MEFs, adenovirus-driven ectopic expression of p21Waf1/Cip1 resulted in a substantial protection against p53-induced apoptosis, indicating that p21Waf1/Cip1 rescued cells from a path of programmed cell death to one of enhanced survival.

AGEMAP: A Gene Expression Database for Aging in Mice

We present the AGEMAP (Atlas of Gene Expression in Mouse Aging Project) gene expression database, which is a resource that catalogs changes in gene expression as a function of age in mice. The AGEMAP database includes expression changes for 8,932 genes in 16 tissues as a function of age. We found great heterogeneity in the amount of transcriptional changes with age in different tissues. Some tissues displayed large transcriptional differences in old mice, suggesting that these tissues may contribute strongly to organismal decline. Other tissues showed few or no changes in expression with age, indicating strong levels of homeostasis throughout life. Based on the pattern of age-related transcriptional changes, we found that tissues could be classified into one of three aging processes: (1) a pattern common to neural tissues, (2) a pattern for vascular tissues, and (3) a pattern for steroid-responsive tissues. We observed that different tissues age in a coordinated fashion in individual mice, such that certain mice exhibit rapid aging, whereas others exhibit slow aging for multiple tissues. Finally, we compared the transcriptional profiles for aging in mice to those from humans, flies, and worms. We found that genes involved in the electron transport chain show common age regulation in all four species, indicating that these genes may be exceptionally good markers of aging. However, we saw no overall correlation of age regulation between mice and humans, suggesting that aging processes in mice and humans may be fundamentally different.

Cell cycle activation linked to neuronal cell death initiated by DNA damage.

Increasing evidence indicates that neurodegeneration involves the activation of the cell cycle machinery in postmitotic neurons. However, the purpose of these cell cycle-associated events in neuronal apoptosis remains unknown. Here we tested the hypothesis that cell cycle activation is a critical component of the DNA damage response in postmitotic neurons. Different genotoxic compounds (etoposide, methotrexate, and homocysteine) induced apoptosis accompanied by cell cycle reentry of terminally differentiated cortical neurons. In contrast, apoptosis initiated by stimuli that do not target DNA (staurosporine and colchicine) did not initiate cell cycle activation. Suppression of the function of ataxia telangiectasia mutated (ATM), a proximal component of DNA damage-induced cell cycle checkpoint pathways, attenuated both apoptosis and cell cycle reentry triggered by DNA damage but did not change the fate of neurons exposed to staurosporine and colchicine. Our data suggest that cell cycle activation is a critical element of the DNA damage response of postmitotic neurons leading to apoptosis.

Posttranscriptional Gene Regulation by Long Noncoding RNA

Eukaryotic cells transcribe a vast number of noncoding RNA species. Among them, long noncoding RNAs (lncRNAs) have been widely implicated in the regulation of gene transcription. However, examples of posttranscriptional gene regulation by lncRNAs are emerging. Through extended base-pairing, lncRNAs can stabilize or promote the translation of target mRNAs, while partial base-pairing facilitates mRNA decay or inhibits target mRNA translation. In the absence of complementarity, lncRNAs can suppress precursor mRNA splicing and translation by acting as decoys of RNA-binding proteins or microRNAs and can compete for microRNA-mediated inhibition leading to increased expression of the mRNA. Through these regulatory mechanisms, lncRNAs can elicit differentiation, proliferation, and cytoprotective programs, underscoring the rising recognition of lncRNA roles in human disease. In this review, we summarize the mechanisms of posttranscriptional gene regulation by lncRNAs identified until now.

microRNA Expression Patterns Reveal Differential Expression of Target Genes with Age

Recent evidence supports a role for microRNAs (miRNAs) in regulating the life span of model organisms. However, little is known about how these small RNAs contribute to human aging. Here, we profiled the expression of over 800 miRNAs in peripheral blood mononuclear cells from young and old individuals by real-time RT-PCR analysis. This genome-wide assessment of miRNA expression revealed that the majority of miRNAs studied decreased in abundance with age. We identified nine miRNAs (miR-103, miR-107, miR-128, miR-130a, miR-155, miR-24, miR-221, miR-496, miR-1538) that were significantly lower in older individuals. Among them, five have been implicated in cancer pathogenesis. Predicted targets of several of these miRNAs, including PI3 kinase (PI3K), c-Kit and H2AX, were found to be elevated with advancing age, supporting a possible role for them in the aging process. Furthermore, we found that decreasing the levels of miR-221 was sufficient to cause a corresponding increase in the expression of the predicted target, PI3K. Taken together, these findings demonstrate that changes in miRNA expression occur with human aging and suggest that miRNAs and their predicted targets have the potential to be diagnostic indicators of age or age-related diseases.