The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.

Anthrax Lethal Toxin (LeTx) demonstrates potent MAPK pathway inhibition and apoptosis in melanoma cells that harbor the activating V600E B-RAF mutation. LeTx is composed of two proteins, PA and LF. Uptake of the toxin into cells is dependent upon proteolytic activation of PA by the ubiquitously expressed furin or furin-like proteases. In order to circumvent nonspecific LeTx activation, a substrate preferably cleaved by gelatinases was substituted for the furin LeTx activation site. Here we have shown the toxicity of this MMP-activated LeTx is dependent on host cell surface MMP-2 and −9 activity as well as the presence of the activating V600E B-RAF mutation, making this toxin dual specific. This additional layer of tumor cell specificity would potentially decrease systemic toxicity from the reduction of nonspecific toxin activation while retaining anti-tumor efficacy in patients with V600E B-RAF melanomas. Moreover, our results indicate that cell surface-associated gelatinase expression can be used to predict sensitivity among V600E B-RAF melanomas. This finding will aid in the better selection of patients that will potentially respond to MMP-activated LeTx therapy.

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor–related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)–deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.

Author Summary

The effects of many pathogenic bacteria are caused by the toxins they release. The toxin released by bacteria that cause anthrax is particularly fascinating since it is made of three different proteins: edema factor, lethal factor, and protective antigen (PA). On their own, each of these proteins is harmless, but when combined, they are deadly. This is because edema factor and lethal factor can exert their poisonous effects only after they have been moved into cells by PA. Determining exactly how PA does this is seen as a critical step in developing medicines that will fight anthrax. That is why a recent report suggesting that LRP6, an outer cell protein, was needed for PA to move the other toxin proteins into cells, was greeted with such interest. However, we now show that mice or cells lacking LRP6, or a related protein called LRP5, are still susceptible to anthrax toxin. The discovery that PA can move lethal factor and edema factor into cells without the help of LRP6 presents a significant challenge to the previously published model. These findings will help focus the efforts of scientists working on new ways to treat anthrax.

While recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.

Glucocorticoids have been shown to play a role in the transforming abilities of human papillomaviruses (HPVs), and glucocorticoid response elements (GREs) have been identified in the upstream regulatory regions (URRs) of various HPV types. These findings have made glucocorticoids potential therapeutic targets for HPV infection. We have previously shown that the URR of HPV type 31 (HPV31) is insensitive to induction by the synthetic glucocorticoid dexamethasone (dex) in monolayer culture, despite the identification of three potential GREs in the 5′ region of the URR. Due to the fact that the HPV life cycle is intimately linked to the differentiation of the host tissue, we chose to determine whether the URR of HPV31 was inducible by dex under differentiating conditions. Upon suspension of cells in a semisolid medium of methylcellulose, we found that the URR of HPV31 was inducible by dex. The three GREs appear to play roles as independent repressors of this inducibility. By 5′ deletion analysis, the element(s) responsible for this induction was localized to nucleotides (nt) 7238 to 7557. Furthermore, we found that the region between nt 7883 and 7900 appears to act as a repressor of dex inducibility. These findings indicate that epithelial differentiation has a profound effect on the action of dex on the URR of HPV31, suggesting that glucocorticoids play an important role in the differentiation-dependent life cycle of HPV.

The upstream regulatory region (URR) of various types of human papillomaviruses (HPVs) has been shown to contain functional glucocorticoid response elements (GREs), including HPV type 11 (HPV11), HPV16, and HPV18. Glucocorticoids have been demonstrated to induce the transcriptional activity of the early promoters of these HPV types. Although it has been assumed that the URR of HPV31 contains at least one GRE, no functionality has been demonstrated. We attempt to show here inducibility of the URR of HPV31 by the synthetic glucocorticoid dexamethasone (dex). By sequence analysis we identified three potential GREs in the URR of HPV31. Gel shift analysis indicated that each of these three sites has the potential to be a functional GRE. However, constructs containing the full-length URR, 5′ deletions of the URR, and an internal fragment of the URR containing all three putative GREs were only weakly inducible by dex. Linker scanning mutants, whereby each potential GRE was replaced individually, in double combination, or in triple combination by a unique polylinker, had no effect on dex inducibility. Replacement of each of the three HPV31 GREs with the GRE of HPV18 failed to induce a response to dex. Placement of the HPV18 GRE into the URR of HPV31 in a region similar to its location in the HPV18 URR was also unable to result in a strong dex induction of the HPV31 URR. These data suggest that the lack of dex inducibility is due to the overall context of the HPV31 URR and may be dependent on the requirements of the major early promoter for transcriptional activation. Finally, replacement of the HPV18 GRE with each of the HPV31 GREs in HPV18 only showed weak inducibility, indicating that the three GREs of HPV31 are in fact only weak inducers of dex. Overall, these data suggest that dex responsiveness, along with oncogenic potential, may provide a possible explanation for the classification of HPV31 as an intermediate-risk virus and demonstrate the complexity of transcriptional regulation of the URR of HPV.

The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.

The function of the 5′ region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5′ region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the viral life cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the viral life cycle. This study identified (i) viral cis elements that regulate transcription in the presence and absence of any viral gene products or viral DNA replication, (ii) the role of host tissue differentiation in viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5′ region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the viral life cycle.