Elizabeth V. Wattenberg

T3 increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells

Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and beta-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase alpha(1)-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.

M1, M3 AND M5 MUSCARINIC RECEPTORS STIMULATE MITOGEN-ACTIVATED PROTEIN KINASE

We report here that the M₁, M₃ and M₅ muscarinic acetylcholine receptor subtypes that have been shown to couple to phosphoinositide hydrolysis also activate the mitogen-activated protein kinase (MAPK). Pharmacological characterization as well as mechanistic details of the activation pathway are presented. Carbachol-induced MAPK activation was time- and concentration-dependent at all subtypes. Pharmacological characterization of the MAPK response revealed that McN-A-343 was a partial agonist at the M₁ and M₃ subtypes, and that pilocarpine was a partial agonist at the M₃ and M₅ receptors. Carbachol-mediated MAPK activation at these receptor subtypes was pertussis toxin and wortmannin insensitive. By contrast, both agents significantly inhibited carbachol-induced MAPK activation by the M₂ muscarinic receptor subtype. Furthermore, two independent single point mutations in the M₁ receptor attenuated carbachol-induced activation of MAPK. Activation of MAPK at the M₁, M₃ and M₅ muscarinic receptor subtypes was not dependent on intracellular or extracellular Ca²⁺, but was partially dependent upon protein kinase C. These data suggest that activation of MAPK by M₁, M₃ and M₅ muscarinic receptors involves protein kinase C-dependent and independent pathways.

Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3

Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

Mixtures of Nickel and Cobalt Chlorides Induce Synergistic Cytotoxic Effects: Implications for Inhalation Exposure Modeling

Workers are often simultaneously exposed to two or more chemicals, yet little is known about the toxicity of most chemical mixtures. The traditional assumption, in the absence of further information, has been that the chemical components of a mixture have mutually independent effects, and the toxic response to multiple chemicals is additive. The data presented here show that mixtures of NiCl(2) and CoCl(2) induce a synergistic (that is, greater than additive) toxic response in cell culture. Immortalized alveolar epithelial type II cells were incubated for 4 h with various concentrations of either NiCl(2), CoCl(2), or NiCl(2) and CoCl(2) together, and cell viability assessed 24 h later. The LD(50) for NiCl(2) was 5.7 mM. CoCl(2), with an LD(50) of 1.1 mM, was about five times more potent than NiCl(2). Mixtures of NiCl(2) and CoCl(2) decreased cell viability synergistically. For example, a mixture of 750 microM NiCl(2) and 750 microM CoCl(2) reduced cell viability by more than three times the value predicted by the additive approach. We used concentration-response data from these studies in a mathematical model; this model describes the equivalent inhalation exposure to an aerosol composed of a mixture of chemicals with different toxicities and also accounts for synergistic responses to these chemicals. Our results along with previous studies using an animal model suggest that these synergisms should be taken into account when conducting future exposure assessments.

Inhibition of mitogen activated protein kinases increases the sensitivity of A549 lung cancer cells to the cytotoxicity induced by a kava chalcone analog

We are interested in investigating the biological activity of chalcones, a major class of compounds found in the beverage kava, in order to develop potent and selective chemopreventive candidates. Consumption of kava in the South Pacific Islands is inversely correlated with cancer incidence, even among smokers. Accordingly, chalcones have anti-cancer activities in animal and cell culture models. To investigate signaling pathways that affect chalcone action we studied a potent analog, (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (chalcone-24). Chalcone-24 was selected from a series of chalcone analogs that were synthesized based on the structures derived from flavokawain compounds found in kava, and screened in A549 lung cancer cells for induction of cytotoxicity and inhibition of NF-κB, a transcription factor associated with cell survival. Incubation of A549 cells with chalcone-24 resulted in a dose-dependent inhibition of cell viability, inhibition of NF-κB, activation of caspases, and activation of extracellular signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK); ERK1/2 and JNK are mitogen activated protein kinases that play central roles in regulating cell fate. Pharmacological inhibitors of ERK1/2 or JNK increased the sensitivity of A549 cells to chalcone-24-induced cytotoxicity, without affecting NF-κB or caspase activity. These results will help refine the synthesis of chalcone analogs to maximize the combination of actions required to prevent and treat cancer.

Modulation of protein kinase signaling cascades by palytoxin.

Although known for its acutely toxic action, palytoxin has also been identified as a type of carcinogenic agent called a tumor promoter. In general tumor promoters do not damage DNA, but instead contribute to carcinogenesis by disrupting the regulation of cellular signaling. The identification of palytoxin as a tumor promoter, together with the recognition that the Na(+), K(+)-ATPase is its receptor, led to research on how palytoxin triggers the modulation of signal transduction pathways. This review focuses on mitogen activated protein (MAP) kinases as mediators of palytoxin-stimulated signaling. MAP kinases are a family of serine/threonine kinases that relay a variety of signals to the cellular machinery that regulates cell fate and function. The studies discussed in this review investigated how palytoxin stimulates MAP kinase activity and, in turn, how MAP kinases mediate the response of cells to palytoxin.

Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction.

Protein prenylation involves the addition of either a farnesyl (C(15)) or geranylgeranyl (C(20)) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.

Photochemical Modulation of Ras-Mediated Signal Transduction Using Caged Farnesyltransferase Inhibitors: Activation by One- and Two-Photon Excitation

The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase is described. The inhibitor, FTI, was caged by alkylation of a critical thiol group with a bromohydroxycoumarin (Bhc) moiety. While Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryl groups. The resulting caged molecule, Bhc‐FTI, can be photolyzed with UV light to release the inhibitor that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc‐FTI can be uncaged by two‐photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies.

Assessment of the Acute and Chronic Health Hazards of Hydraulic Fracturing Fluids

There is growing concern about how hydraulic fracturing affects public health because this activity involves handling large volumes of fluids that contain toxic and carcinogenic constituents, which are injected under high pressure through wells into the subsurface to release oil and gas from tight shale formations. The constituents of hydraulic fracturing fluids (HFFs) present occupational health risks because workers may be directly exposed to them, and general public health risks because of potential air and water contamination. Hazard identification, which focuses on the types of toxicity that substances may cause, is an important step in the complex health risk assessment of hydraulic fracturing. This article presents a practical and adaptable tool for the hazard identification of HFF constituents, and its use in the analysis of HFF constituents reported to be used in 2,850 wells in North Dakota between December 2009 and November 2013. Of the 569 reported constituents, 347 could be identified by a Chemical Abstract Service Registration Number (CASRN) and matching constituent name. The remainder could not be identified either because of trade secret labeling (210) or because of an invalid CASRN (12). Eleven public databases were searched for health hazard information on thirteen health hazard endpoints for 168 identifiable constituents that had at least 25 reports of use. Health hazard counts were generated for chronic and acute endpoints, including those associated with oral, inhalation, ocular, and dermal exposure. Eleven of the constituents listed in the top 30 by total health hazard count were also listed in the top 30 by reports of use. This includes naphthalene, which along with benzyl chloride, has the highest health hazard count. The top 25 constituents reportedly used in North Dakota largely overlap with those reported for Texas and Pennsylvania, despite different geologic formations, target resources (oil vs. gas), and disclosure requirements. Altogether, this database provides a public health tool to help inform stakeholders about potential health hazards, and to aid in the reformulation of less hazardous HFFs.

Investigation of the sequence and length dependence for cell-penetrating prenylated peptides.

Cell penetrating peptides are useful delivery tools for introducing molecules of interest into cells. A new class of cell penetrating molecules has been recently reported-cell penetrating, prenylated peptides. In this study a series of such peptides was synthesized to examine the relationship between peptide sequence and level of peptide internalization and to probe their mechanism of internalization. This study revealed that prenylated peptides internalize via a non-endocytotic pathway regardless of sequence. Sequence length and identity was found to play a role in peptide uptake but prenylated sequences as short as two amino acids were found to exhibit significant cell penetrating properties.