Increase In C-fos Protein Levels Research Articles

Activation of AP-1 By JAK2V617F through Induction and Stabilization of c-FOS

Abstract JAK2V617F, a gain-of-function mutation in the tyrosine kinase JAK2, has been detected in most patients with Philadelphia chromosome (Ph)-negative classical myeloproliferative neoplasms (MPNs). The identification of the JAK2 mutation in MPNs led to the development of JAK2 inhibitors, which suppress the symptoms of this disease. However, treatment with these inhibitors rarely leads to molecular remission and is associated with side effects caused by blockade of function of wild-type JAK2. In addition, resistance has been reported; therefore, a new therapeutic strategy based on understanding of MPN development is required. Here, we found that JAK2V617F dramatically upregulates cytomegalovirus (CMV) promoter activity in HEK293T cells. By performing a reporter assay using luciferase, we found that a non-canonical AP-1 binding motif is responsible for this activation of the CMV promoter. By performing a series of biochemical analyses, including an electrophoresis mobility shift assay and a supershift assay with antibodies, we found that c-FOS, a member of the AP-1 family of proteins, bound to oligo duplex DNA containing the identified motif only when JAK2V617F was expressed. A reporter construct harboring the identified sequence alone showed a robust increase in reporter assay when JAK2V617F was induced, while mutation of the AP-1 binding motif abolished the response to JAK2V617F. The results showed that c-FOS overexpression alone is sufficient for activation of the promoter containing the non-canonical AP-1 binding site, demonstrating that c-FOS is a downstream target of JAK2V617F in CMV promoter activation. The activation of this reporter construct was also observed in leukemia cell lines UT-7/EPO and UT-7/GM when JAK2V617F is expressed, which implies that this activation is not limited to HEK293T cells. We found that the activation of AP-1 by JAK2V617F was partly due to robust induction of c-FOS mRNA (41 fold) followed by increase in c-FOS protein levels. In addition, we found that, the ubiquitin-proteasome protein degradation pathway responsible for c-FOS degradation was perturbed in HEK293T cells expressing JAK2V617F, which at least partly contributed to c-FOS protein accumulation and subsequent AP-1 activation. Multiple inhibitors of known downstream pathways of JAK2, such as the mitogen activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways, were analyzed using the reporter assay. The results showed that none of them suppressed JAK2V617F-dependent reporter activation. Furthermore, inhibition of the STAT pathway by expression of the dominant-negative form of STAT1, STAT3, or STAT5 failed to suppress AP-1 activation by JAK2V617F. Consistent with this observation, expression of the active form of STAT1, STAT3, and STAT5 did not affect AP-1 activity. Taken together, we conclude that AP-1 activation by JAK2V617F through c-FOS induction involves an unidentified molecular mechanism. Although our observation is limited to an in vitro system, our finding suggests that deregulation of AP-1 plays a role in MPN development. The AP-1 reporter assay system that we have established should be useful not only for screening novel compounds for MPN treatment but also in studying the novel pathway regulated by JAK2V617F, which would lead to the development of a novel therapeutic strategy against MPNs. Disclosures No relevant conflicts of interest to declare.

Calsenilin interacts with transcriptional co‐repressor C‐terminal binding protein(s)

Calsenilin/potassium channel-interacting protein (KChIP)3/ downstream regulatory element sequence antagonist modulator (DREAM) is a neuronal calcium-binding protein that has been shown to have multiple functions in the cell, including the regulation of presenilin processing, repression of transcription and modulation of A-type potassium channels. To gain a better understanding of the precise role of calsenilin in specific cellular compartments, an interactor hunt for proteins that bind to the N-terminal domain of calsenilin was carried out. Using a yeast two-hybrid system and co-immunoprecipitation studies, we have identified the transcriptional co-repressor C-terminal binding protein (CtBP)2 as an interactor for calsenilin and have shown that the two proteins can interact in vivo. In co-immunoprecipitation studies, calsenilin also interacted with CtBP1, a CtBP2 homolog. Our data also showed a calsenilin-dependent increase in c-fos protein levels in CtBP knockout fibroblasts, suggesting that CtBP may modulate the transcriptional repression of c-fos by calsenilin. Furthermore, the finding that histone deacetylase protein and activity were associated with the calsenilin-CtBP immunocomplex suggests a mechanism by which calsenilin-CtBP may act to repress transcription. Finally, we demonstrated that calsenilin and CtBP are present in synaptic vesicles and can interact in vivo.

Extracellular Signal-Regulated Kinase Transmits Palytoxin-Stimulated Signals Leading to Altered Gene Expression in Mouse Keratinocytes

We have been probing the molecular mechanisms of tumor promoters that stimulate distinct initial signals to define critical downstream biochemical events in carcinogenesis. The action of the novel skin tumor promoter palytoxin on signaling and gene expression in keratinocytes, the primary target cells of tumor promoters, was therefore investigated. Palytoxin stimulated an increase in mRNA for matrix metalloproteinase-13 (MMP-13), an enzyme implicated in carcinogenesis, in a keratinocyte cell line derived from initiated mouse skin (308). Palytoxin stimulated an increase in c-Fos binding to the activator protein-1 (AP-1) site present in the promoter of the mouse MMP-13 gene. This effect was specific because palytoxin had little effect on c-Jun, JunB, JunD, FosB, Fra-1, or Fra-2 binding or on overall levels of transcription factor binding. The increase in c-Fos binding corresponded to a palytoxin-stimulated increase in c-Fos protein levels. Palytoxin stimulated the activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase, and p38. The MAPK kinase inhibitor PD 98059 blocked palytoxin-stimulated ERK activation. PD 98059 also blocked the palytoxin-stimulated increases in c-Fos protein levels, c-Fos binding to the AP-1 site, and MMP-13 mRNA. These studies identify important differences between palytoxin-stimulated signaling in keratinocytes derived from initiated mouse skin, the biologically relevant cell type, and other cell lines. Specifically, our data suggest that, in keratinocytes derived from initiated mouse skin, ERK plays an important role in transmitting palytoxin-stimulated signals to three downstream targets that are likely to affect carcinogenesis: c-Fos, AP-1, and MMP-13.

Induction in human osteoblastic cells (SaOS2) of the early response genes fos, jun, and myc by the amino terminal fragment (ATF) of urokinase.

Previous studies have demonstrated that overexpression of urinary plasminogen activator (uPA) in rat prostate cancer cells results in increased skeletal metastases, which are primarily of the osteoblastic variety. The osseous activation induced by the metastases appears to be mediated through the amino terminal fragment (ATF) of uPA, which lacks the catalytic domain and can act as a growth factor for osteoblasts. To explore further the mechanism of action of uPA in bone cells, we evaluated the effects of ATF on modulating the expression of various proto-oncogenes. Human-osteoblast-derived osteosarcoma cells, SaOS2, were treated with graded doses of ATF for 10-120 min, and effects on early response proto-oncogenes were monitored. ATF increased c-myc, c-jun, and c-fos gene expression in a time-dependent manner for up to 60 min, after which mRNA levels fell. The maximum induction was seen in c-fos gene expression, which was found to be dose dependent. This effect of ATF was localized to its growth-factorlike domain. Examination of the half life of these transcripts in the presence of the transcriptional inhibitor actinomycin D demonstrated that ATF does not alter the stability of c-fos mRNA in these bone cells. Nuclear run-off assays indicated that ATF effects were due to stimulation of c-fos gene transcription. An increase in c-fos protein levels was correlated with the augmentation of its mRNA in ATF-treated SaOS2 cells. Pretreatment of SaOS2 cells with the protein tyrosine kinase inhibitor herbimycin and recombinant soluble uPA receptor (uPAR) caused a significant reduction in the ability of ATF to induce c-fos expression. These results demonstrate a novel role for uPA in activating early response proto-oncogenes, in particular c-fos, which plays an important role in bone cell growth and differentiation and may be a key factor in the signal transduction pathway of ATF.