Karina Krotova

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca(2+)-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect l-[(3)H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC.

Hypoxic upregulation of arginase II in human lung endothelial cells.

Activated arginase has been implicated in many diseases including cancer, immune cell dysfunction, infections, and vascular disease. Enhanced arginase activity has been reported in lungs of patients with pulmonary artery hypertension. We used hypoxia as a model for pulmonary hypertension and studied the effect of exposure to hypoxia on arginase activity in human lung microvascular endothelial cells (HMVEC). Hypoxia induces upregulation of arginase activity as well as mRNA and protein levels of arginase II (Arg II), the only arginase isoform we were able to identify in HMVEC. In endothelial cells, arginase shares and competes for the substrate l-arginine with nitric oxide (NO) synthase (NOS). Through regulation of substrate availability for NOS, arginase is able to modulate NO production. To evaluate the role of Arg II in regulation of NO production under hypoxia, we compared NO output (RFL-6 reporter assay) in cells with normal and silenced Arg II. Exposure to hypoxia led to an increase in NO levels produced by HMVEC. Inhibition of Arg II by specific small interfering RNA or by the pharmacological inhibitor BEC additionally enhanced the levels of NO. Another possible role for activated arginase is involvement in regulation of cell proliferation. However, we showed that hypoxia decreased cell proliferation and upregulated Arg II did not have an effect on cell proliferation. Since hypoxia-inducible factors (HIF) are a family of transcriptional factors activated by hypoxia, we tested the possibility of involvement of HIF-1 and HIF-2 in regulation of Arg II under hypoxia. The silencing of HIF-2 but not HIF-1 prevented the activation of Arg II by hypoxia.

Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation

1 Myristoylated pseudosubstrate of PKCζ (mPS) – a synthetic myristoylated peptide with a sequence (13 amino acids) mimicking the endogenous PKCζ pseudosubstrate region – is considered a selective cell‐permeable inhibitor of PKCζ. We present strong evidence that in endothelial cells the action of mPS is not limited to inhibition of PKC activity and that myristoylation of certain peptides can activate eNOS (endothelial nitric oxide synthase) through Akt phosphorylation. 2 mPS at micromolar concentrations (1–10 μM) induced profound phosphorylation of eNOS, Akt, ERK 1/2, and p38 MAPK in cultured pulmonary artery endothelial cells (PAEC). The same changes were observed after treatment of PAEC with a myristoylated scrambled version of mPS (mScr), whereas a cell‐permeable version of PKCζ pseudosubstrate fused to the HIV‐TAT membrane‐translocating peptide did not induce analogous changes, suggesting that myristoylation confers new properties on the peptides consisting of activation of different signaling pathways in endothelial cells. 3 In addition to mPS and mScr, a number of other myristoylated peptides induced phosphorylation of eNOS suggesting that myristoylation of peptides can activate eNOS by mechanisms unrelated to inhibition of PKC. All active myristoylated peptides contained basic amino acids motif and were longer than six amino acids. 4 Activation of eNOS by myristoylated peptides was dependent on the PI3K/Akt pathway and the rise of intracellular calcium and was associated with an elevation of cGMP levels in PAEC and with relaxation of precontracted isolated pulmonary artery segments. 5 Myristoylated peptides can be considered a new class of activators of NO production in endothelial cells and that using mPS as a specific inhibitor of PKCζ should be done with caution, especially in endothelial cells.

Endothelial arginase II responds to pharmacological inhibition by elevation in protein level

Arginase is an enzyme which converts arginine to ornithine and urea. Recently, arginase has been implicated in many physiological and pathological processes including vascular diseases. Inhibition of arginase activity by pharmacological inhibitors is a useful tool to study the biology of arginases and their possible role in therapy. There are several arginase-specific inhibitors commercially available. Herein, we show that some of these inhibitors lead to an increase in arginase II protein level and activity. These effects should be anticipated when these inhibitors are in use or during the testing of new arginase inhibitors.

Erdj3 Has an Essential Role for Z Variant Alpha-1-Antitrypsin Degradation

Alpha‐1‐antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at amino acid 342 in the mature protein, resulting in the Z mutation of the alpha‐1‐antitrypsin gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes and monocytes, causing a toxic gain of function. Retained ZAAT is eliminated by ER‐associated degradation and autophagy. We hypothesized that alpha‐1‐antitrypsin (AAT)‐interacting proteins play critical roles in quality control of human AAT. Using co‐immunoprecipitation, we identified ERdj3, an ER‐resident Hsp40 family member, as a part of the AAT trafficking network. Depleting ERdj3 increased the rate of ZAAT degradation in hepatocytes by redirecting ZAAT to the ER calreticulin‐EDEM1 pathway, followed by autophagosome formation. In the Huh7.5 cell line, ZAAT ER clearance resulted from enhancing ERdj3‐mediated ZAAT degradation by silencing ERdj3 while simultaneously enhancing autophagy. In this context, ERdj3 suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD‐related liver disease. J. Cell. Biochem. 118: 3090–3101, 2017.

Alpha-1 Antitrypsin-Deficient Macrophages Have Increased Matriptase-Mediated Proteolytic Activity

&NA; Alpha‐1 antitrypsin (AAT) deficiency‐associated emphysema is largely attributed to insufficient inhibition of neutrophil elastase released from neutrophils. Correcting AAT levels using augmentation therapy only slows disease progression, and that suggests a more complex process of lung destruction. Because alveolar macrophages (M&phis;) express AAT, we propose that the expression and intracellular accumulation of mutated Z‐AAT (the most common mutation) compromises M&phis; function and contributes to emphysema development. Extracellular matrix (ECM) degradation is a hallmark of emphysema pathology. In this study, M&phis; from individuals with Z‐AAT (Z‐M&phis;) have greater proteolytic activity on ECM than do normal M&phis;. This abnormal Z‐M&phis; activity is not abrogated by supplementation with exogenous AAT and is likely the result of cellular dysfunction induced by intracellular accumulation of Z‐AAT. Using pharmacologic inhibitors, we show that several classes of proteases are involved in matrix degradation by Z‐M&phis;. Importantly, compared with normal M&phis;, the membrane‐bound serine protease, matriptase, is present in Z‐M&phis; at higher levels and contributes to their proteolytic activity on ECM. In addition, we identified matrix metalloproteinase (MMP)‐14, a membrane‐anchored metalloproteinase, as a novel substrate for matriptase, and showed that matriptase regulates the levels of MMP‐14 on the cell surface. Thus, high levels of matriptase may contribute to increased ECM degradation by Z‐M&phis;, both directly and through MMP‐14 activation. In summary, the expression of Z‐AAT in M&phis; confers increased proteolytic activity on ECM. This proteolytic activity is not rescued by exogenous AAT supplementation and could thus contribute to augmentation resistance in AAT deficiency‐associated emphysema.

SVIP regulates Z variant alpha-1 antitrypsin retro-translocation by inhibiting ubiquitin ligase gp78

Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder characterized by early-onset emphysema and liver disease. The most common disease-causing mutation is a single amino acid substitution (Glu/Lys) at amino acid 342 of the mature protein, resulting in disruption of the 290–342 salt bridge (an electrophoretic abnormality defining the mutation [Z allele, or ZAAT]), protein misfolding, polymerization, and accumulation in the endoplasmic reticulum of hepatocytes and monocytes. The Z allele causes a toxic gain of function, and the E3 ubiquitin ligase gp78 promotes degradation and increased solubility of endogenous ZAAT. We hypothesized that the accumulation of ZAAT is influenced by modulation of gp78 E3 ligase and SVIP (small VCP-interacting protein) interaction with p97/VCP in ZAAT-expressing hepatocytes. We showed that the SVIP inhibitory effect on ERAD due to overexpression causes the accumulation of ZAAT in a human Z hepatocyte–like cell line (AT01). Overexpression of gp78, as well as SVIP suppression, induces gp78-VCP/p97 interaction in AT01 cells. This interaction leads to retro-translocation of ZAAT and reduction of the SVIP inhibitory role in ERAD. In this context, overexpression of gp78 or SVIP suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD.

Abstract B248: Antitumor activity mediated by N-myristoylated cationic peptides (myrCP).

Although new chemotherapeutic agents are improving the survival rate of cancer patients, all of them have serious toxic side effects. Thus, it is still necessary to search for new agents with minimal side effects or to enhance the effectiveness of known cytotoxic agents at a reduced dose. The modification of peptides by adding myristol group is commonly used to facilitate intracellular delivery. It has been shown recently that the combination of myristoylation and the cationic charge of peptide confer new biological activity to the peptide. We examined the possibility of using such peptides for the treatment of cancer. In present studies, we evaluated the effect of two N-myristoylated cationic peptides (myrCP) on several human cancer cell lines: H1299 (lung), MCF7 (breast), and PC3 (prostate). The first peptide mimics the endogenous Protein kinase C (PKC) pseudosubstrate region and it is considered as a selective inhibitor of PKC (myr-SIYRRGARRWRKL-OH). This peptide has a dual effect on cancer cells as it prevents proliferation by inhibiting PKC and also serves as a cytotoxic agent. The second peptide that is a scrambled version of the first peptide (myr-RLYRKRIWRSAGR-OH) was used as a control to ensure that cytotoxic effect is not only dependent on PKC inhibition. We found that both myrCP had superior cytotoxic effect on cancer cells. Moreover, they dramatically enhanced the activity of commonly used chemotherapeutic agents such as mitoxantrone and cisplatin which were active in combination with peptides at significantly lower concentration. Since the lipophilic nature of myristoylation facilitates penetration of these peptides across cell membranes, we analyzed what lipids can be involved in such interaction. Membrane lipid arrays showed that myrCP preferably binds to PtdIns (3, 4, 5) P3, phosphatidic acid and at less extent to PI (4, 5) P2. Our results suggested that lipid membrane composition of cells could play an essential role in efficacy of myrCP. In summary, the potential outcome of the current studies will be the development of a useful therapeutic tool for efficient killing of tumor cells. Considering that cytotoxic effects of myrCP are non-specific to the type of cancer, a similar strategy can be applied for many different malignancies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B248. Citation Format: Karina Krotova, George Aslanidi. Antitumor activity mediated by N-myristoylated cationic peptides (myrCP). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B248.