Jonathan Weitzman

JunD protects cells from p53-dependent senescence and apoptosis

JunD is the most broadly expressed member of the Jun family and the AP-1 transcription factor complex. Primary fibroblasts lacking JunD displayed p53-dependent growth arrest, upregulated p19(Arf) expression, and premature senescence. In contrast, immortalized cell lines lacking JunD showed increased proliferation and higher cyclinD1 levels. These properties are reminiscent of the effects of oncogenic Ras expression on primary and established cell cultures. Furthermore, JunD(-/-) fibroblasts exhibited increased p53-dependent apoptosis upon ultraviolet irradiation and were sensitive to the cytotoxic effects of TNF-alpha. The antiapoptotic role of JunD was confirmed using an in vivo model of TNF-mediated hepatitis. We propose that JunD protects cells from senescence, or apoptotic responses to stress stimuli, by acting as a modulator of the signaling pathways that link Ras to p53.

Transcription factor JunD, deprived of menin, switches from growth suppressor to growth promoter

Different components of the AP1 transcription factor complex appear to have distinct effects on cell proliferation and transformation. In contrast to other AP1 components, JunD has been shown to inhibit cell proliferation. Also, in prior studies, JunD alone bound menin, product of the MEN1 tumor suppressor gene, and JunDs transcriptional activity was inhibited by menin, suggesting that JunD might achieve all or most of its unique properties through binding to menin. Analyses of JunD and menin effects on proliferation, morphology, and cyclin D1 in stable cell lines unmasked an unexpected growth promoting activity of JunD. Whereas stable overexpression of wild-type (wt) mouse JunD in JunD–/– immortalized fibroblasts inhibited their proliferation and reverted their transformed-like phenotype, overexpression of a missense mouse JunD mutant (mJunDG42E) with disabled binding to menin showed opposite or growth promoting effects. Similarly, stable overexpression of wt mouse JunD in wt immortalized fibroblasts inhibited growth. In contrast, its overexpression in Men1–/– immortalized fibroblasts enhanced their already transformed-like characteristics. To conclude, JunD changed from growth suppressor to growth promoter when its binding to menin was prevented by a JunD mutant unable to bind menin or by Men1-null genetic background.

Functional cooperation between JunD and NF-kappaB in rat hepatocytes.

AP-1 and NF-κB are rapidly activated during liver regeneration. Whether these parallel inductions have potential functional implications is not known. Isolated rat hepatocytes were stimulated with two mitogens, epidermal growth factor or hepatocyte growth factor and with tumor necrosis factor α, a cytokine involved in the liver regenerative response in vivo and a strong inducer of NF-κB. All three cytokines increased AP-1 and NF-κB binding to their cognate cis-element and induced a 2.5-fold activation of NF-κB-dependent transcription. Inactivation of AP-1 by TAM67, a dominant negative mutant of AP-1 drastically inhibited basal and cytokine-induced NF-κB transactivation. Overexpression of Jun D, but not of the other Jun or Fos proteins increased by threefold NF-κB transactivation. Functional cooperation between JunD and p65 was demonstrated in a simple Gal-hybrid system. Finally, a twofold decrease in NF-κB transactivation was found in hepatocytes isolated from JunD−/− mice compared with hepatocytes from JunD+/+ mice. Altogether these data demonstrate a functional cooperation of p65 with JunD, a major constituent of AP-1 in normal hepatocytes.

AP-1 dimers regulate transcription of the p14/p19ARF tumor suppressor gene.

Evidence is accumulating about the role of individual AP-1 components in cell proliferation and transformation. Notably, Ras-mediated transformation is characterized by the upregulation of particular AP-1 members, such as c-Jun and Fra-1. The p14/p19ARF tumor suppressor gene is a key link between oncogenic Ras signaling and the p53 pathway. We explored the involvement of AP-1 dimers in the transcriptional regulation of the p14/p19ARF gene. We demonstrate that both the human and mouse ARF promoters are transcriptional targets of selective AP-1 dimers. The ARF promoter is regulated specifically by AP-1 heterodimers containing Fra-1. Overexpression of c-Jun∼Fra-1 dimers in primary murine fibroblast cells led to the upregulation of the endogenous ARF protein and growth arrest. Conversely, inhibition of c-Jun or Fra-1 protein levels resulted in decreased ARF expression. In addition, we show that AP-1 dimers cooperate with oncogenic Ras in the transcriptional activation of the p14/p19ARF promoter. Thus, AP-1 heterodimers may contribute to the regulation of ARF expression upon oncogenic signaling.

Fra‐1 promotes growth and survival in RAS‐transformed thyroid cells by controlling cyclin A transcription

Fra‐1 is frequently overexpressed in epithelial cancers and implicated in invasiveness. We previously showed that Fra‐1 plays crucial roles in RAS transformation in rat thyroid cells and mouse fibroblasts. Here, we report a novel role for Fra‐1 as a regulator of mitotic progression in RAS‐transformed thyroid cells. Fra‐1 expression and phosphorylation are regulated during the cell cycle, peaking at G2/M. Knockdown of Fra‐1 caused a proliferative block and apoptosis. Although most Fra‐1‐knockdown cells accumulated in G2, a fraction of cells entering M‐phase underwent abortive cell division and exhibited hallmarks of genomic instability (micronuclei, lagging chromosomes and anaphase bridges). Furthermore, we established a link between Fra‐1 and the cell‐cycle machinery by identifying cyclin A as a novel transcriptional target of Fra‐1. During the cell cycle, Fra‐1 was recruited to the cyclin A gene (ccna2) promoter, binding to previously unidentified AP‐1 sites and the CRE. Fra‐1 also induced the expression of JunB, which in turn interacts with the cyclin A promoter. Hence, Fra‐1 induction is important in thyroid tumorigenesis, critically regulating cyclin expression and cell‐cycle progression.

The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria

Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c‐Jun N‐terminal kinase (JNK) and the AP‐1 transcription factor. We found that JNK/AP‐1 activation is associated with elevated levels of Rab11 protein in Theileria‐transformed B cells. We show that AP‐1 regulates rab11a promoter activity in B cells and that the induction of c‐Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP‐1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP‐1 pathway during cell transformation might assure that the regulation of recycling endosomes is co‐ordinated with cell‐cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A).

Eyeing-up stem cell transplantation

The recent identification of neural stem cells has encouraged scientists that these undifferentiated progenitor cells might hold the key to treatment of many neuro-degenerative disorders. This possibility took a step forward when researchers led by Don Sakaguchi (Iowa State University, IA) and Michael Young Harvard Medical School, MA, USA) successfully transplanted and integrated stem cells into the eyes of postnatal Brazillian opossums. Spheres of green-fluorescent protein (GFP) expressing rat hippocampal-derived or embryonic mouse stem cells were injected into the eyes of opossum pups. Immunoreactivity for several neural and retinal markers was observed, indicating that these transplanted cells had integrated into the eye and differentiated into retinal neurons. Integration and survival of these cells raises the prospect of treatment for human retinopathies such as glaucoma and macular degeneration. (AR)

Life and death in the JUNgle

Experiments with transgenic and knockout mice have begun to elucidate distinct roles for the three members of the Jun family of transcription factors. Mice with tissue-specific loss of JunB develop a myeloproliferative disorder, emphasizing the important roles that Jun proteins play in regulating life and death decisions in disease.

Bax and p53 are differentially involved in the regulation of caspase-3 expression and activation during neurodegeneration in Lurcher mice.

Intrinsic Purkinje cell death in heterozygous Lurcher (Grid2Lc/+) mice is accompanied by the target-related death of granule cells and olivary neurons. The expression of pro-caspase-3 is increased in Grid2Lc/+ Purkinje cells and activated caspase-3 is detected in all three cell types before their death. Bax inactivation in Grid2Lc/+ mutants rescues granule cells but not Purkinje cells. Here, we show that, while Bax inactivation inhibits caspase-3 activation in both cell types, p53 inactivation does not affect caspase-3 activation and neuronal loss in Grid2Lc/+ mice. The up-regulation of pro-caspase-3 in Grid2Lc/+ Purkinje cells is Bax and p53 independent. These results suggest that Grid2Lc/+ granule cell death is dependent on Bax and caspase-3 activation, whereas several pathways can mediate Grid2Lc/+ Purkinje cell death.

p16Ink4a and p19Arf: terrible twins

p16Ink4a and p19Arf might be considered to be molecular twins. They are born from the same genetic locus CDKN2A (INK4a/ARF) and have tricked biologists for some years about which one is important in preventing cancer. Now cancer biologists have nailed down this terrible twosome and formally proved that both are crucial for inhibiting tumorigenesis. The best way to ‘prove’ that a gene is a tumour suppressor is to mutate the gene in the mouse and show subsequent tumour susceptibility. So, although it will not come as a surprise to learn that mice lacking p16Ink4a form tumours, it is a relief to cancer biologists to have p16Ink4a officially declared a tumour suppressor gene together with his infamous twin p19Arf (1xLoss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Krimpenfort, P. et al. Nature. 2001; 413: 83–86Crossref | PubMed | Scopus (394)See all References, 2xLoss of p16Ink4a with retention of p19Arf predispose mice to tumorigenesis. Sharpless, N.E. et al. Nature. 2001; 413: 86–91Crossref | PubMed | Scopus (549)See all References).The two sibling proteins are encoded at one of the most remarkable loci in the human genome – one short stretch of DNA that generates two overlapping transcripts that encode two structurally unrelated proteins, both of which inhibit the cell cycle. p16Ink4a inhibits cyclin-dependent kinases CDK4 and CDK6, thereby regulating the retinoblastoma pRB pathway. By contrast, p19Arf (called p14Arf in humans) achieves cell-cycle arrest by stabilizing the p53 protein. This is biology at its most elegant. Given their ability to regulate both pRb and p53 checkpoint pathways, its hardly surprising that the CDK2A-INK4A/ARF locus is mutated, or silenced epigenetically, in many human tumours. Although some of these mutations knock out both proteins, others affect just one of the pair, implying that both might behave as independent tumour suppressors. Researchers have turned to genetically engineered mice to solve the riddle of the tumour suppressor twins.Several years ago Kamijo et al.3xTumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Kamijo, T. et al. Cell. 1997; 91: 649–659Abstract | Full Text | Full Text PDF | PubMedSee all References3 showed that mice lacking p19Arf develop tumours early in life, demonstrating that p19Arf is a bona fide tumour suppressor. Now two independent groups (Krimpenfort et al.1xLoss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Krimpenfort, P. et al. Nature. 2001; 413: 83–86Crossref | PubMed | Scopus (394)See all References1 and Sharpless et al.2xLoss of p16Ink4a with retention of p19Arf predispose mice to tumorigenesis. Sharpless, N.E. et al. Nature. 2001; 413: 86–91Crossref | PubMed | Scopus (549)See all References2) have engineered mice with mutations so that they express normal p19Arf but no p16Ink4a. These mutant animals developed normally and the adult mice are quite perky, except for some slight increases in thymocyte numbers. Surprisingly, fibroblasts derived from these animals behaved quite normally too, in terms of proliferation, senescence or Ras-transformation. This is in marked contrast to the abnormal growth of fibroblasts lacking p19Arf.But as the p16Ink4a-null mice grew older, some of them developed tumours including soft-tissue sarcomas, lymphomas and melanomas. When the researchers looked at carcinogen-induced malignancy, both groups found that the mutation dramatically affected susceptibility. Mice treated with the carcinogen 7,12-dimethylbenzanthracene (DMBA) developed lung adenomas, soft-tissue carcinomas, skin papillomas and melanomas. Melanomas are often found in human patients with germline mutations in the CDKN2A locus. Furthermore, the researchers found that combining mutations in p19Arf and p16Ink4a leads to an increase in the number and range of spontaneous tumours. These studies offer an animal model to explore the role of the CDKN2A locus in human malignancies. Twins tend to get blamed as a pair. It looks like each of these talented twins plays a distinct role in tumour suppression.