Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function.

Mitochondria are increasingly recognized as key mediators of acute cellular stress responses in asthma. However, the distinct roles of regulators of mitochondrial physiology on allergic asthma phenotypes are currently unknown. The mitochondrial Ca2+ uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca2+ uptake into the mitochondrial matrix. To understand the function of MCU in models of allergic asthma, in vitro and in vivo studies were performed using models of functional deficiency or knockout of MCU. In primary human respiratory epithelial cells, MCU inhibition abrogated mitochondrial Ca2+ uptake and reactive oxygen species (ROS) production, preserved the mitochondrial membrane potential and protected from apoptosis in response to the pleiotropic Th2 cytokine IL-13. Consequently, epithelial barrier function was maintained with MCU inhibition. Similarly, the endothelial barrier was preserved in respiratory epithelium isolated from MCU-/- mice after exposure to IL-13. In the ovalbumin-model of allergic airway disease, MCU deficiency resulted in decreased apoptosis within the large airway epithelial cells. Concordantly, expression of the tight junction protein ZO-1 was preserved, indicative of maintenance of epithelial barrier function. These data implicate mitochondrial Ca2+ uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.

Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma.

Excessive ROS promote allergic asthma, a condition characterized by airway inflammation, eosinophilic inflammation, and increased airway hyperreactivity (AHR). The mechanisms by which airway ROS are increased and the relationship between increased airway ROS and disease phenotypes are incompletely defined. Mitochondria are an important source of cellular ROS production, and our group discovered that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is present in mitochondria and activated by oxidation. Furthermore, mitochondrial-targeted antioxidant therapy reduced the severity of allergic asthma in a mouse model. Based on these findings, we developed a mouse model of CaMKII inhibition targeted to mitochondria in airway epithelium. We challenged these mice with OVA or Aspergillus fumigatus. Mitochondrial CaMKII inhibition abrogated AHR, inflammation, and eosinophilia following OVA and A. fumigatus challenge. Mitochondrial ROS were decreased after agonist stimulation in the presence of mitochondrial CaMKII inhibition. This correlated with blunted induction of NF-κB, the NLRP3 inflammasome, and eosinophilia in transgenic mice. These findings demonstrate a pivotal role for mitochondrial CaMKII in airway epithelium in mitochondrial ROS generation, eosinophilic inflammation, and AHR, providing insights into how mitochondrial ROS mediate features of allergic asthma.

CaMKII inhibition in type II pneumocytes protects from bleomycin-induced pulmonary fibrosis by preventing Ca2+-dependent apoptosis.

The calcium and calmodulin-dependent kinase II (CaMKII) translates increases in intracellular Ca(2+) into downstream signaling events. Its function in pulmonary pathologies remains largely unknown. CaMKII is a well-known mediator of apoptosis and regulator of endoplasmic reticulum (ER) Ca(2+). ER stress and apoptosis of type II pneumocytes lead to aberrant tissue repair and progressive collagen deposition in pulmonary fibrosis. Thus we hypothesized that CaMKII inhibition alleviates fibrosis in response to bleomycin by attenuating apoptosis and ER stress of type II pneumocytes. We first established that CaMKII was strongly expressed in the distal respiratory epithelium, in particular in surfactant protein-C-positive type II pneumocytes, and activated after bleomycin instillation. We generated a novel transgenic model of inducible expression of the CaMKII inhibitor peptide AC3-I limited to type II pneumocytes (Tg SPC-AC3-I). Tg SPC-AC3-I mice were protected from development of pulmonary fibrosis after bleomycin exposure compared with wild-type mice. CaMKII inhibition also provided protection from apoptosis in type II pneumocytes in vitro and in vivo. Moreover, intracellular Ca(2+) levels and ER stress were increased by bleomycin and significantly blunted with CaMKII inhibition in vitro. These data demonstrate that CaMKII inhibition prevents type II pneumocyte apoptosis and development of pulmonary fibrosis in response to bleomycin. CaMKII inhibition may therefore be a promising approach to prevent or ameliorate the progression of pulmonary fibrosis.

NEDD4 ubiquitin ligase is a putative oncogene in endometrial cancer that activates IGF-1R/PI3K/Akt signaling.

OBJECTIVE: The PI3K/Akt pathway is frequently dysregulated in endometrial cancer, the most common gynecologic malignancy. Emerging evidence identifies the ubiquitin ligase NEDD4 as a key regulator of the PI3K/Akt pathway via activation of insulin-like growth factor-1 receptor (IGF-1R). Our objective was to understand the role of NEDD4 in endometrial cancer. METHODS: NEDD4 expression was assessed by immunohistochemistry in a tissue microarray with 77 endometrial lesions ranging from normal benign endometrium to tumor specimens of varying stage and grade. Studies were extended to a panel of eight endometrial cancer cell lines phenotypically representing the most common endometrial patient tumors. RESULTS: Immunohistochemistry demonstrated robust staining of NEDD4 in endometrial tumor specimens, with greater NEDD4 expression in the most aggressive tumors. Expression of NEDD4 was detected in a majority of endometrial cancer cell lines surveyed. Exogenous overexpression of murine Nedd4 in endometrial cancer cell lines with modest endogenous NEDD4 expression resulted in a significant increase in the rate of proliferation. Nedd4 overexpression also promoted an increase in cell surface localization of IGF-1R and activation of Akt. Inhibition of PI3K/Akt signaling reversed the enhanced cell growth in Nedd4-overexpressing endometrial cancer cells. In addition, the expression of NEDD4 in endometrial tumors positively correlated with the Akt downstream effector FoxM1. CONCLUSIONS: This study identifies NEDD4 as a putative oncogene in endometrial cancer that may augment activation of the IGF-1R/PI3K/Akt signaling pathway.

Mitochondrial-targeted antioxidant therapy decreases transforming growth factor-ß-mediated collagen production in a murine asthma model.

Asthma is a disease of acute and chronic inflammation in which cytokines play a critical role in orchestrating the allergic inflammatory response. IL-13 and transforming growth factor (TGF)-ß promote fibrotic airway remodeling, a major contributor to disease severity. Improved understanding is needed, because current therapies are inadequate for suppressing development of airway fibrosis. IL-13 is known to stimulate respiratory epithelial cells to produce TGF-ß, but the mechanism through which this occurs is unknown. Here, we tested the hypothesis that reactive oxygen species (ROS) are a critical signaling intermediary between IL-13 or allergen stimulation and TGF-ß-dependent airway remodeling. We used cultured human bronchial epithelial cells and an in vivo mouse model of allergic asthma to map a pathway where allergens enhanced mitochondrial ROS, which is an essential upstream signal for TGF-ß activation and enhanced collagen production and deposition in airway fibroblasts. We show that mitochondria in airway epithelium are an essential source of ROS that activate TGF-ß expression and activity. TGF-ß from airway epithelium stimulates collagen expression in fibroblasts, contributing to an early fibrotic response to allergen exposure in cultured human airway cells and in ovalbumin-challenged mice. Treatment with the mitochondrial-targeted antioxidant, (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), significantly attenuated mitochondrial ROS, TGF-ß, and collagen deposition in OVA-challenged mice and in cultured human epithelial cells. Our findings suggest that mitochondria are a critical source of ROS for promoting TGF-ß activity that contributes to airway remodeling in allergic asthma. Mitochondrial-targeted antioxidants may be a novel approach for future asthma therapies.

Epigenetic modification restores functional PR expression in endometrial cancer cells.

Endometrial cancer, the most common gynecologic malignancy, is a hormonally-regulated tumor. Response to progestin-based therapy correlates positively with progesterone receptor (PR) expression. However, many endometrial tumors have low levels or loss of PR, limiting the clinical application of progestin. We evaluated the ability of epigenetic modulators to restore functional PR expression in Type I endometrial cancer cells with low basal PR. Treatment with the histone deacetylase inhibitor (HDACi) LBH589 induced a profound upregulation of PR mRNA. LBH589 restored PR protein expression at 24 hours and sustained expression for 72 hours, even in the presence of progesterone. LBH589 promoted a dose-dependent increase in PR protein levels, with an obvious increase with 10 nM LBH589. To investigate if the restored PR is functional as a transcription factor, we examined PR nuclear localization and expression of PRE- or Sp1-containing target genes. After treatment with LBH589 in the absence or presence of progesterone, PR nuclear expression was increased as demonstrated by Western blotting of nuclear fractions and immunostaining. Next, restored PR upregulated FoxO1, p21, and p27 and downregulated cyclin D1 in a ligand-dependent manner. Finally, LBH589 treatment induced cell cycle arrest in G1 that was further augmented by progesterone. Regulation of PR target genes was also achieved with other HDAC inhibitors, indicating that agents in this class work similarly with respect to PR. Our findings reveal that epigenetic modulators can restore endogenous functional PR expression in endometrial cancer cells and suggest that strategies to re-establish PR expression will resensitize endometrial tumors to progestin therapy.

Cl- channels in basolateral TAL membranes. XVI. MTAL and CTAL cells each contain the mRNAs encoding mmClC-Ka and mcClC-Ka.

BACKGROUND: Our prior data indicate that two separate but homologous basolateral chloride (Cl-) channels, mmClC-Ka and mcClC-Ka, are the principal mediators of net Cl- absorption in mouse medullary thick ascending limb (MTAL) and cortical thick ascending limb (CTAL) cells, respectively. In the present studies, we evaluated the possibility that there might be translational or post-translational suppression of mmClC-Ka and mcClC-Ka activity in CTAL and MTAL cells, respectively. METHODS: Polymerase chain reaction (PCR) fragments were prepared that were highly specific for either mmClC-Ka or mcClC-Ka, the cDNAs encoding mmClC-Ka and mcClC-Ka, respectively. RESULTS: Using reverse transcription (RT)-PCR with these highly specific products, mRNAs specific for non-homologous channel sequences in either mmClC-Ka or mcClC-Ka were present in both MTAL and CTAL cells. CONCLUSIONS: Both mouse MTAL and CTAL cells contain the mRNAs encoding mmClC-Ka and mcClC-Ka. There may be translational or post-translational suppression of mmClC-Ka activity in CTAL cells, and of mcClC-Ka activity in MTAL cells.