c-jun is essential for sympathetic neuronal death induced by NGF withdrawal but not by p75 activation.

Sympathetic neurons depend on NGF binding to TrkA for their survival during vertebrate development. NGF deprivation initiates a transcription-dependent apoptotic response, which is suggested to require activation of the transcription factor c-Jun. Similarly, apoptosis can also be induced by selective activation of the p75 neurotrophin receptor. The transcriptional dependency of p75-mediated cell death has not been determined; however, c-Jun NH2-terminal kinase has been implicated as an essential component. Because the c-jun-null mutation is early embryonic lethal, thereby hindering a genetic analysis, we used the Cre-lox system to conditionally delete this gene. Sympathetic neurons isolated from postnatal day 1 c-jun-floxed mice were infected with an adenovirus expressing Cre recombinase or GFP and analyzed for their dependence on NGF for survival. Cre immunopositive neurons survived NGF withdrawal, whereas those expressing GFP or those uninfected underwent apoptosis within 48 h, as determined by DAPI staining. In contrast, brain-derived neurotrophic factor (BDNF) binding to p75 resulted in an equivalent level of apoptosis in neurons expressing Cre, GFP, and uninfected cells. Nevertheless, cycloheximide treatment prevented BDNF-mediated apoptosis. These results indicate that whereas c-jun is required for apoptosis in sympathetic neurons on NGF withdrawal, an alternate signaling pathway must be induced on p75 activation.

AP-1: one switch for many signals.

The transcription factor AP-1 is activated in response to an incredible array of stimuli, including mitogenic growth factors, inflammatory cytokines, growth factors of the TGF-beta family, UV and ionizing irradiation, cellular stress, antigen binding, and neoplastic transformation. In this review, I discuss genetic evidence that supports a role for AP-1 in the cellular response to some of these stimuli and describe biochemical properties that might explain the ability of this transcription factor to activate different sets of genes in response to different stimuli.

c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms.

c-Jun is a component of the transcription factor AP-1, which is activated by a wide variety of extracellular stimuli. The regulation of c-Jun is complex and involves both increases in the levels of c-Jun protein as well as phosphorylation of specific serines (63 and 73) by Jun N-terminal kinase (JNK). We have used fibroblasts derived from c-Jun null embryos to define the role of c-Jun in two separate processes: cell growth and apoptosis. We show that in fibroblasts, c-Jun is required for progression through the G1 phase of the cell cycle; c-Jun-mediated G1 progression occurs by a mechanism that involves direct transcriptional control of the cyclin D1 gene, establishing a molecular link between growth factor signaling and cell cycle regulators. In addition, c-Jun protects cells from UV-induced cell death and cooperates with NF-kappaB to prevent apoptosis induced by tumor necrosis factor alpha (TNFalpha). c-Jun mediated G1 progression is independent of phosphorylation of serines 63/73; however, protection from apoptosis in response to UV, a potent inducer of JNK/SAP kinase activity, requires serines 63/73. The results reveal critical roles for c-Jun in two different cellular processes and show that different extracellular stimuli can target c-Jun by distinct biochemical mechanisms.

Physical and genetic interactions between Alx4 and Cart1.

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, we have analyzed the functional and genetic relationships between Alx4 and Cart1. The two proteins have similar DNA-binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions. First, mutation of Cart1 exacerbates Alx4-dependent polydactyly in a manner that is dependent on gene dosage. Second, there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures. Third, double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development.

Transcriptional activation and transformation by FosB protein require phosphorylation of the carboxyl-terminal activation domain.

The transcription factor AP-1, composed of Fos-Jun dimers, mediates some aspects of the cellular response to growth factors. Transcriptional activation and neoplastic transformation by FosB, a member of the Fos family of proteins, require the presence of a potent C-terminal activation domain. Here we show by mutational analysis that the FosB C-terminal domain has a proline-based motif that is essential for both of these functions. Phosphopeptide mapping experiments show that the C terminus of FosB is phosphorylated within a cluster of functionally redundant serine residues that is adjacent to this proline-based motif. Mutation of these serine residues to alanine severely reduces the ability of this region to function as an activation domain and inhibits the ability of FosB protein to function as a transforming protein. Several observations suggest that the kinase responsible for phosphorylation of these sites is distinct from the mitogen-activation protein kinases and stress-activated protein kinases. Our results show that transcriptional activation and neoplastic transformation by the FosB protein are dependent on phosphorylation within the C terminus. This form of control may provide a potential mechanism of signal integration at the level of a single transcription factor.

Cellular transformation and malignancy induced by ras require c-jun.

ras is an important oncogene in experimental animals and humans. In addition, activated ras proteins are potent inducers of the transcription factor AP-1, which is composed of heterodimeric complexes of Fos and Jun proteins. Together with the fact that deregulated expression of some AP-1 proteins can cause neoplastic transformation, this finding suggests that AP-1 may function as a critical ras effector. We have tested this hypothesis directly by analyzing the response to activated ras in cells that harbor a null mutation in the c-jun gene. The transcriptional response of AP-1-responsive genes to activated ras is severely impaired in c-jun null fibroblasts. Compared with wild-type cells, the c-jun null cells lack many characteristics of ras transformation, including loss of contact inhibition, anchorage independence, and tumorigenicity in nude mice; these properties are restored by forced expression of c-jun. Rare tumorigenic variants of ras-expressing c-jun null fibroblasts do arise. Analysis of these variants reveals a consistent restoration of AP-1 activity. The results provide genetic evidence that c-jun is a crucial effector for transformation by activated ras proteins.

Proto-oncogene FosB: the amino terminus encodes a regulatory function required for transformation.

Overexpression of some members of the Fos gene family, including FosB, leads to transformation of established rodent fibroblasts. We have previously shown that transformation by FosB requires the presence of a C-terminal transcriptional activation domain. We now report that transformation by FosB also requires an intact DNA-binding domain composed of the functionally bipartite basic region and leucine zipper as well as sequences present in the N terminus that serve a regulatory function. Deletion of the N-terminal sequences results in proteins impaired in transcriptional activation and transformation. This region does not itself function as a transcriptional activation domain but instead regulates the transactivation functions present in the FosB-Jun complex. The requirement for this N-terminal region can be abolished by the presence of a strong constitutive activation domain. The primary sequence of the region that we have defined is highly conserved in the Fos family of proteins, suggesting functional conservation.

Transformation by FosB requires a trans-activation domain missing in FosB2 that can be substituted by heterologous activation domains.

Two functionally distinct proteins derived from the FosB gene by alternative splicing have recently been described. FosB protein transforms fibroblasts efficiently, whereas FosB2 protein, a carboxy-terminally truncated form of FosB, does not, despite the fact that both proteins can participate in high-affinity, sequence-specific DNA binding as part of a heterodimeric complex with c-Jun protein. We show here that the functional difference between these proteins is the result of the presence of a potent proline-rich transcriptional activation domain in the carboxy-terminal amino acids unique to FosB. This conclusion is supported by three lines of evidence: (1) Mutations in the carboxy-terminal region of FosB that impair transcriptional activation also reduce transforming potential, despite the fact that DNA binding as part of a complex with c-Jun is not affected; (2) the carboxy-terminal region unique to FosB functions as an activation domain when fused to the DNA-binding domain of GAL4; and (3) transforming potential can be conferred on FosB2 by fusing any of several different well-characterized trans-activation domains. These results identify an additional functional requirement for transformation by Fos proteins and have implications for the mechanism(s) of mitogenic signaling by the AP-1 transcription complex.

An alternative spliced form of FosB is a negative regulator of transcriptional activation and transformation by Fos proteins.

Two forms of FosB transcript and their products can be identified in mouse NIH 3T3 cells following serum induction. The larger RNA codes for a 338-amino acid protein, whereas the smaller RNA results from the removal of an additional 140 nucleotides from FosB mRNA by alternative splicing. This alternative splicing event places a stop codon following the "leucine zipper" region and results in a shorter protein (FosB2) of 237 amino acids that lacks 101 amino acids at the carboxyl terminus. FosB2 is able to form heterodimers with c-Jun and bind to an AP-1 site but is not able to activate the transcription of promoters containing AP-1 sites. Furthermore, FosB2 can not only suppress the transcriptional activation by c-Fos and c-Jun of promoters containing an AP-1 site but also interferes with the transforming potential of viral and cellular Fos proteins. We propose that FosB2 protein functions as a trans-negative regulator.

The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors.

The stability of certain mRNAs is known to be affected by translation. Some mRNAs appear to be protected from rapid degradation by translation, whereas degradation is coupled to translation for other mRNAs. The molecular determinants of this selective effect of translation are unknown. One example of this effect is the induction of early-response gene mRNAs in the presence of translation inhibitors. To define the molecular basis of induction of early-response gene mRNA expression by inhibitors of protein synthesis, we have performed a mutational analysis of one member of the early response gene family, the c-myc gene. We find that induction by cycloheximide is due to stabilization of c-myc transcripts. The requirements for increased expression of c-myc mRNA by cycloheximide are the presence of the sequence encoding c-myc amino acids 335-439 on a mRNA that can be translated; all other portions of the c-myc gene are dispensable, and this sequence can confer induction of mRNA expression by protein synthesis inhibitors on a heterologous gene. By direct measurement of mRNA turnover in the absence of transcription-blocking drugs, we show that this sequence can function as a selective mRNA destabilizing element, that turnover mediated by this element is translation dependent, and turnover mediated by this element is inhibited by actinomycin D. Our results support the hypothesis that degradation of c-myc mRNA is coupled to translation, that the sequences specifying this form of degradation are contained in the protein-coding sequence, and that translation inhibitors induce expression of c-myc mRNA by blocking turnover mediated by this element.

Translation of c-myc mRNA is required for its post-transcriptional regulation during myogenesis.

During the differentiation of C2 myoblasts into differentiated myotubes, there is a marked decrease in the abundance of c-myc mRNA. c-myc transcription initiation and elongation do not change significantly, but the turnover of c-myc transcripts appears to be accelerated during differentiation. We examined the expression of several recombinant c-myc genes introduced into C2 cells by stable transfection and found that mRNAs containing the c-myc protein-coding region of exons 2 and 3 are appropriately regulated; the upstream c-myc sequences, the long leader sequence encoded by c-myc exon 1, and the 3' untranslated region are dispensible for proper regulation. Regulation appears to affect c-myc transcripts that are bound to polyribosomes, and the mRNA from a gene with a point mutation in the translation initiation codon is no longer properly regulated. We conclude that down-regulation of c-myc expression during myogenesis is post-transcriptional and requires the presence of sequences encoding the c-myc protein in a form that can be translated.

Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant.

Phenotypic revertants of Finkel-Biskis-Riley (FBR)-murine sarcoma virus-transformed rat fibroblasts were isolated on the basis of their adherence to plastic tissue culture dishes in the absence of divalent cations. Some revertants had sustained deletions or inactivating mutations of the v-fos gene. However, two revertants expressed a functional v-fos gene at levels equal to that in the transformed parental cells, and therefore phenotypic reversion was due to mutations in nonviral genes. These revertants were considered nontransformed according to four criteria: (i) they were flat and had a nontransformed morphology, (ii) they were contact inhibited when grown to confluence, (iii) they did not display anchorage-independent growth in soft agar, and (iv) they did not form tumors in nude mice. Somatic-cell hybrids between the revertants and the transformed parental cells were nontransformed, suggesting that the revertants had sustained an activating mutation of a gene capable of suppressing transformation. The expression of c-jun, junB, and junD was not altered in the revertants, and they could not be transformed by transfection with a c-jun expression vector. The revertants were resistant to transformation by an activated c-Ha-ras gene but were susceptible to transformation by simian virus 40. Our results demonstrate the existence of a class of revertants that harbor genes capable of suppressing transformation by v-fos and some other oncogenes. This contrasts with previously described revertants of transformation by v-fos that contain recessive mutations.

Verapamil restoration of daunorubicin responsiveness in daunorubicin-resistant Ehrlich ascites carcinoma.

We have studied the influence of verapamil hydrochloride on the in vitro and in vivo effects of daunorubicin in Ehrlich ascites carcinoma. Daunorubicin-sensitive tumor was rendered resistant to daunorubicin by the continuous treatment of sequential generations of tumor-bearing BALB/c mice. The ability of daunorubicin to inhibit [(3)H]uridine and [(3)H]thymidine incorporation and the effect of daunorubicin on the mean survival time of host animals bearing daunorubicin-sensitive and daunorubicin-resistant Ehrlich ascites carcinoma were compared. The addition of verapamil to daunorubicin in vitro reduced the concentration of daunorubicin required to inhibit 50% of DNA and RNA synthesis in the daunorubicin-resistant tumor to that required in the daunorubicin-sensitive tumor, from 6 and 4.4 mug/ml to 1.5 and 1.3 mug/ml, respectively. Verapamil also restored drug sensitivity to daunorubicin-resistant Ehrlich ascites carcinoma in vivo. The 21.7+/-0.7 d mean survival time (MST) of BALB/c mice bearing daunorubicin-resistant tumor treated with daunorubicin alone rose to 44.0+/-0.7 d when the same tumor was treated with verapamil and daunorubicin, P < 0.001. This in vivo effect is specific for daunorubicin-resistant Ehrlich ascites carcinoma, since there is no alteration in MST of BALB/c mice bearing daunorubicin-sensitive or daunorubicin-resistant tumor when they are treated with verapamil alone or when BALB/c mice bearing daunorubicin-sensitive tumor are treated with daunorubicin and verapamil.