Robert F. Hennigan

Pseudomonas aeruginosa Anaerobic Respiration in Biofilms: Relationships to Cystic Fibrosis Pathogenesis

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

Transcription factors control invasion: AP-1 the first among equals

Metastasis, the aggressive spread of a malignant tumor to distant organs, is a major cause of death in cancer patients. Despite this critical role in cancer outcomes, the molecular mechanisms that control this process are just beginning to be understood. Metastasis is largely dependent upon the ability of tumor cells to invade the barrier formed by the basement membrane and to migrate through neighboring tissues. This review will summarize the evidence that tumor cell invasion is the result of oncogene-mediated signal transduction pathways that control the expression of a specific set of genes that together mediate tumor cell invasion. We focus on the role of the transcription factor AP-1 to both induce the expression of genes that function as invasion effectors and repress other genes that function as invasion suppressors. This identifies AP-1 as a critical regulator of a complex program of gene expression that defines the invasive phenotype.

Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.

Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways.

The Polo-like kinases (Plks) are a conserved family of kinases that contribute to cell cycle regulation, particularly in G2 and mitosis. In mammals, there are at least three members of the Plk family. Here we show that Plk3 is a stress response protein that becomes phosphorylated following DNA damage or mitotic spindle disruption. Phosphorylation enhances its kinase activity and is dependent upon ataxia telangiectasia-mutated (ATM) in the former case but not the latter. Plk3 associates with complexes of multiple sizes ranging from 150 to greater then 600 kDa. In its unphosphorylated form it elutes from a sizing column at about 400 kDa whereas it associates with complexes of 150 and 600 kDa when phosphorylated. Among the proteins with which it physically associates and utilizes, as substrates are Chk2 and P53. It phosphorylates Chk2 on a residue different from threonine 68 (Thr68), the principal target for ATM. While ATM is necessary for phosphorylation and activation of Chk2 in vivo, Plk3 seems to contribute to its full activation. In its phosphorylated form it also coelutes and forms a complex with unpolymerized tubulin. In aggregate, the data argue that Plk3 is a multifunctional protein that associates with multiple complexes and that contributes to response to stress incurred by DNA damage and mitotic spindle disruption, albeit via different pathways.

CD44 associates with EGFR and erbB2 in metastasizing mammary carcinoma cells.

Type I receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) and erbB2, have been implicated in mammary carcinoma growth and metastasis. Recent evidence suggests that type I receptor signaling may be mediated by the CD44 family of transmembrane glycoproteins that also have been implicated in mammary tumor progression. Here, the authors tested whether CD44, EGFR, and erbB2 interacted and colocalized with one another in four mammary carcinoma cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and MDA-MB-436) and in cytology samples obtained from patients with metastatic breast cancer. CD44 constitutively colocalized and coimmunoprecipitated with erbB2 and EGFR in all four mammary carcinoma cell lines. CD44 also colocalized with erbB2 and EGFR in all cytology samples expressing erbB2. CD44 colocalized with EGFR in cells from only 1 of 16 erbB2-negative cytology samples. These data indicate that CD44-EGFR-erbB2 protein complexes occur in a high proportion of metastatic mammary carcinomas and suggest that CD44-type I receptor colocalization may be a novel prognostic marker for aggressive mammary cancers.

Two-pronged survival strategy for the major cystic fibrosis pathogen, Pseudomonas aeruginosa, lacking the capacity to degrade nitric oxide during anaerobic respiration.

Protection from NO gas, a toxic byproduct of anaerobic respiration in Pseudomonas aeruginosa, is mediated by nitric oxide (NO) reductase (NOR), the norCB gene product. Nevertheless, a norCB mutant that accumulated ∼13.6 μM NO paradoxically survived anaerobic growth. Transcription of genes encoding nitrate and nitrite reductases, the enzymes responsible for NO production, was reduced >50‐ and 2.5‐fold in the norCB mutant. This was due, in part, to a predicted compromise of the [4Fe–4S]2+ cluster in the anaerobic regulator ANR by physiological NO levels, resulting in an inability to bind to its cognate promoter DNA sequences. Remarkably, two O2‐dependent dioxygenases, homogentisate‐1,2‐dioxygenase (HmgA) and 4‐hydroxyphenylpyruvate dioxygenase (Hpd), were derepressed in the norCB mutant. Electron paramagnetic resonance studies showed that HmgA and Hpd bound NO avidly, and helped protect the norCB mutant in anaerobic biofilms. These data suggest that protection of a P. aeruginosa norCB mutant against anaerobic NO toxicity occurs by both control of NO supply and reassignment of metabolic enzymes to the task of NO sequestration.

Critical regulation of genes for tumor cell migration by AP-1

The AP-1 transcription factor plays a critical role in regulating tumor cell proliferation and has been implicated in controlling a program of gene expression that mediates cell motility and invasion in vitro. We have utilized two dominant negative AP-1 constructs, TAM67 and aFos, each fused to GFP, to investigate the role of AP-1 complexes in an invasive, clinically derived human tumor cell line, HT-1080. As expected, high levels of both GFP-TAM67 and GFP-aFos arrested HT-1080 cells in the G1 phase of the cell cycle. Strikingly, at low levels GFP-aFos, but not GFP-TAM67, caused a change in colony morphology, impairment of directional motility in a monolayer wound healing assay, as well as inhibition of chemotaxis and haptotaxis. Microarray analysis identified a novel set of AP-1 target genes, including the tumor suppressor TSCL-1 and regulators of actin cytoskeletal dynamics, including the gelsolin-like actin capping protein CapG. The demonstration that AP-1 regulates the expression of genes involved in tumor cell motility and cytoskeletal dynamics in a clinically derived human tumor cell line identifies new pathways of control for tumor cell motility.

NF2 -deficient cells depend on the Rac1-canonical Wnt signaling pathway to promote the loss of contact inhibition of proliferation

The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a membrane/cytoskeleton protein necessary for the maintenance of contact inhibition of growth in cells. Bi-allelic inactivation of NF2 is known to cause multiple cancers in both humans and mice. However, the mechanism through which merlin exerts its tumor-suppressive function remains obscure. In this report, we show that NF2 knockout mouse embryonic fibroblasts lost contact inhibition of cell proliferation and contained significantly increased canonical Wnt signaling. Inhibition of Rac1, the activity of which is inversely regulated by NF2, through the use of a dominant-negative mutant, small hairpin RNA or a small molecule inhibitor in NF2-deficient cells, was able to suppress elevated Wnt signals as shown by reduced activity of the T-cell factor 4 (TCF4) transcription factor. Dominant-negative TCF4 or Rac1 mutant, as well as a small molecule inhibition of Wnt, were able to curb NF2 deficiency-elicited cell proliferation at the confluent state. Thus, Rac1-mediated canonical Wnt signaling is essential for the loss of contact inhibition in NF2-deficient cells.

Retinoblastoma Tumor Suppressor: Analyses of Dynamic Behavior in Living Cells Reveal Multiple Modes of Regulation

ABSTRACT The retinoblastoma tumor suppressor, RB, assembles multiprotein complexes to mediate cell cycle inhibition. Although many RB binding partners have been suggested to underlie these functions, the validity of these interactions on the behavior of RB complexes in living cells has not been investigated. Here, we studied the dynamic behavior of RB by using green fluorescent protein-RB fusion proteins. Although these proteins were universally nuclear, phosphorylation or oncoprotein binding mediated their active exclusion from the nucleolus. In vivo imaging approaches revealed that RB exists in dynamic equilibrium between a highly mobile and a slower diffusing species, and genetic lesions associated with tumorigenesis increased the fraction of RB in a highly mobile state. The RB complexes dictating cell cycle arrest were surprisingly dynamic and harbored a relatively short residence time on chromatin. In contrast, this rapid exchange was attenuated in cells that are hypersensitive to RB, suggesting that responsiveness may inversely correlate with mobility. The stability of RB dynamics within the cell was additionally modified by the presence and function of critical corepressors. Last, the RB-assembled complexes present in living cells were primarily associated with E2F binding sites in chromatin. In contrast to RB, E2F1 consistently maintained a stable association with E2F sites regardless of cell type. Together, these results elucidate the kinetic framework of RB tumor suppressor action in transcriptional repression and cell cycle regulation.

Fluorescence Resonance Energy Transfer Analysis of Merlin Conformational Changes

ABSTRACT Neurofibromatosis type 2 is an inherited autosomal disorder caused by biallelic inactivation of the NF2 tumor suppressor gene. The NF2 gene encodes a 70-kDa protein, merlin, which is a member of the ezrin-radixin-moesin (ERM) family. ERM proteins are believed to be regulated by a transition between a closed conformation, formed by binding of their N-terminal FERM domain and C-terminal tail domain (CTD), and an open conformation, in which the two domains do not interact. Previous work suggests that the tumor suppressor function of merlin is similarly regulated and that only the closed form is active. Therefore, understanding the mechanisms that control its conformation is crucial. We have developed a series of probes that measures merlin conformation by fluorescence resonance energy transfer, both as purified protein and in live cells. Using these tools, we find that merlin exists predominately as a monomer in a stable, closed conformation that is mediated by the central α-helical domain. The contribution from the FERM-CTD interaction to the closed conformation appears to be less important. Upon phosphorylation or interaction with an effector protein, merlin undergoes a subtle conformational change, suggesting a novel mechanism that modulates the interaction between the FERM domain and the CTD.