Proapoptotic protein Bim attenuates estrogen-enhanced survival in lymphangioleiomyomatosis

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Abstract

Lymphangioleiomyomatosis (LAM) is a progressive lung disease that primarily affects young women. Genetic evidence suggests that LAM cells bearing TSC2 mutations migrate to the lungs, proliferate, and cause cystic remodeling. The female predominance indicates that estrogen plays a critical role in LAM pathogenesis, and we have proposed that estrogen promotes LAM cell metastasis by inhibition of anoikis. We report here that estrogen increased LAM patient–derived cells’ resistance to anoikis in vitro, accompanied by decreased accumulation of the proapoptotic protein Bim, an activator of anoikis. The resistance to anoikis was reversed by the proteasome inhibitor, bortezomib. Treatment of LAM patient–derived cells with estrogen plus bortezomib promoted anoikis compared with estrogen alone. Depletion of Bim by siRNA in TSC2-deficient cells resulted in anoikis resistance. Treatment of mice with bortezomib reduced estrogen-promoted lung colonization of TSC2-deficient cells. Importantly, molecular depletion of Bim by siRNA in Tsc2-deficient cells increased lung colonization in a mouse model. Collectively, these data indicate that Bim plays a key role in estrogen-enhanced survival of LAM patient–derived cells under detached conditions that occur with dissemination. Thus, targeting Bim may be a plausible future treatment strategy in patients with LAM.

Authors

Chenggang Li, Na Li, Xiaolei Liu, Erik Y. Zhang, Yang Sun, Kouhei Masuda, Jing Li, Julia Sun, Tasha Morrison, Xiangke Li, Yuanguang Chen, Jiang Wang, Nagla A. Karim, Yi Zhang, John Blenis, Mauricio J. Reginato, Elizabeth P. Henske, Jane J. Yu

SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion

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Abstract

The repulsive guidance cue SLIT2 and its receptor ROBO2 are required for kidney development and podocyte foot process structure, but the SLIT2/ROBO2 signaling mechanism regulating podocyte function is not known. Here we report that a potentially novel signaling pathway consisting of SLIT/ROBO Rho GTPase activating protein 1 (SRGAP1) and nonmuscle myosin IIA (NMIIA) regulates podocyte adhesion downstream of ROBO2. We found that the myosin II regulatory light chain (MRLC), a subunit of NMIIA, interacts directly with SRGAP1 and forms a complex with ROBO2/SRGAP1/NMIIA in the presence of SLIT2. Immunostaining demonstrated that SRGAP1 is a podocyte protein and is colocalized with ROBO2 on the basal surface of podocytes. In addition, SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen. In vivo studies further showed that podocyte-specific knockout of Robo2 protects mice from hypertension-induced podocyte detachment and albuminuria and also partially rescues the podocyte-loss phenotype in Myh9 knockout mice. Thus, we have identified SLIT2/ROBO2/SRGAP1/NMIIA as a potentially novel signaling pathway in kidney podocytes, which may play a role in regulating podocyte adhesion and attachment. Our findings also suggest that SLIT2/ROBO2 signaling might be a therapeutic target for kidney diseases associated with podocyte detachment and loss.

Authors

Xueping Fan, Hongying Yang, Sudhir Kumar, Kathleen E. Tumelty, Anna Pisarek-Horowitz, Hila Milo Rasouly, Richa Sharma, Stefanie Chan, Edyta Tyminski, Michael Shamashkin, Mostafa Belghasem, Joel M. Henderson, Anthony J. Coyle, David J. Salant, Stephen P. Berasi, Weining Lu

Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism

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Abstract

Cullin-3 (CUL3) mutations (CUL3Δ9) were previously identified in hypertensive patients with pseudohypoaldosteronism type-II (PHAII), but the mechanism causing hypertension and whether this is driven by renal tubular or extratubular mechanisms remains unknown. We report that selective expression of CUL3Δ9 in smooth muscle acts by interfering with expression and function of endogenous CUL3, resulting in impaired turnover of the CUL3 substrate RhoA, increased RhoA activity, and augmented RhoA/Rho kinase signaling. This caused vascular dysfunction and increased arterial pressure under baseline conditions and a marked increase in arterial pressure, collagen deposition, and vascular stiffness in response to a subpressor dose of angiotensin II, which did not cause hypertension in control mice. Inhibition of total cullin activity increased the level of CUL3 substrates cyclin E and RhoA, and expression of CUL3Δ9 decreased the level of the active form of endogenous CUL3 in human aortic smooth muscle cells. These data indicate that selective expression of the Cul3Δ9 mutation in vascular smooth muscle phenocopies the hypertension observed in Cul3Δ9 human subjects and suggest that mutations in CUL3 cause human hypertension in part through a mechanism involving smooth muscle dysfunction initiated by a loss of CUL3-mediated degradation of RhoA.

Authors

Larry N. Agbor, Stella-Rita C. Ibeawuchi, Chunyan Hu, Jing Wu, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund

Epigenetic regulation of macrophage polarization and inflammation by DNA methylation in obesity

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Abstract

Obesity is associated with increased classically activated M1 adipose tissue macrophages (ATMs) and decreased alternatively activated M2 ATMs, both of which contribute to obesity-induced inflammation and insulin resistance. However, the underlying mechanism remains unclear. We find that inhibiting DNA methylation pharmacologically using 5-aza-2′-deoxycytidine or genetically by DNA methyltransferase 1 (DNMT1) deletion promotes alternative activation and suppresses inflammation in macrophages. Consistently, mice with myeloid DNMT1 deficiency exhibit enhanced macrophage alternative activation, suppressed macrophage inflammation, and are protected from obesity-induced inflammation and insulin resistance. The promoter and 5′-untranslated region of peroxisome proliferator-activated receptor γ1 (PPARγ1) are enriched with CpGs and are epigenetically regulated. The saturated fatty acids stearate and palmitate and the inflammatory cytokine TNF-α significantly increase, whereas the TH2 cytokine IL-4 significantly decreases PPARγ1 promoter DNA methylation. Accordingly, inhibiting PPARγ1 promoter DNA methylation pharmacologically using 5-aza-2′-deoxycytidine or genetically by DNMT1 deletion promotes macrophage alternative activation. Our data therefore establish DNA hypermethylation at the PPARγ1 promoter induced by obesity-related factors as a critical determinant of ATM proinflammatory activation and inflammation, which contributes to insulin resistance in obesity.

Authors

Xianfeng Wang, Qiang Cao, Liqing Yu, Huidong Shi, Bingzhong Xue, Hang Shi

Longitudinal PET imaging demonstrates biphasic CAR T cell responses in survivors

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Abstract

Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.

Authors

Yogindra Vedvyas, Enda Shevlin, Marjan Zaman, Irene M. Min, Alejandro Amor-Coarasa, Spencer Park, Susan Park, Keon-Woo Kwon, Turner Smith, Yonghua Luo, Dohyun Kim, Young Kim, Benedict Law, Richard Ting, John Babich, Moonsoo M. Jin

ML372 blocks SMN ubiquitination and improves spinal muscular atrophy pathology in mice

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Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. SMA results from insufficient levels of the survival motor neuron (SMN) protein, and studies in animal models of the disease have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient–derived cells. We show here that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice.

Authors

Mahlet B. Abera, Jingbo Xiao, Jonathan Nofziger, Steve Titus, Noel Southall, Wei Zheng, Kasey E. Moritz, Marc Ferrer, Jonathan J. Cherry, Elliot J. Androphy, Amy Wang, Xin Xu, Christopher Austin, Kenneth H. Fischbeck, Juan J. Marugan, Barrington G. Burnett

Bcl-2–associated athanogene 3 protects the heart from ischemia/reperfusion injury

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Abstract

Bcl-2–associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9–expressing (rAAV9-expressing) BAG3 or GFP and subjected to I/R. To elucidate molecular mechanisms by which BAG3 protects against I/R injury, neonatal mouse ventricular cardiomyocytes (NMVCs) in which BAG3 levels were modified by adenovirus expressing (Ad-expressing) BAG3 or siBAG3 were exposed to hypoxia/reoxygenation (H/R). H/R significantly reduced NMVC BAG3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux. The deleterious effects of H/R on apoptosis and autophagy were recapitulated by knockdown of BAG3 with Ad-siBAG3 and were rescued by Ad-BAG3. In vivo, treatment of mice with rAAV9-BAG3 prior to I/R significantly decreased infarct size and improved left ventricular function when compared with mice receiving rAAV9-GFP and improved markers of autophagy and apoptosis. These findings suggest that BAG3 may provide a therapeutic target in patients undergoing reperfusion after myocardial infarction.

Authors

Feifei Su, Valerie D. Myers, Tijana Knezevic, JuFang Wang, Erhe Gao, Muniswamy Madesh, Farzaneh G. Tahrir, Manish K. Gupta, Jennifer Gordon, Joseph Rabinowitz, Frederick V. Ramsey, Douglas G. Tilley, Kamel Khalili, Joseph Y. Cheung, Arthur M. Feldman

Quantitative evaluation of the antiretroviral efficacy of dolutegravir

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Abstract

The second-generation HIV-1 integrase strand transfer inhibitor (InSTI) dolutegravir (DTG) has had a major impact on the treatment of HIV-1 infection. Here we describe important but previously undetermined pharmacodynamic parameters for DTG. We show that the dose-response curve slope, which indicates cooperativity and is a major determinant of antiviral activity, is higher for DTG than for first-generation InSTIs. This steepness does not reflect inhibition of multiple steps in the HIV-1 life cycle, as is the case for allosteric integrase inhibitors and HIV-1 protease inhibitors. We also show that degree of independence, a metric of interaction favorability between antiretroviral drugs, is high for DTG and nucleoside reverse transcriptase inhibitors. Finally, we demonstrate poor selective advantage for HIV-1 bearing InSTI resistance mutations. Selective advantage, which incorporates both the magnitude of resistance conferred by a mutation and its fitness cost, explains the high genetic barrier to DTG resistance. Together, these parameters provide an explanation for the remarkable clinical success of DTG.

Authors

Sarah B. Laskey, Robert F. Siliciano

Humanized neuronal chimeric mouse brain generated by neonatally engrafted human iPSC-derived primitive neural progenitor cells

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Abstract

The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However, generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)–derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons, which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology, we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure, function, and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders.

Authors

Chen Chen, Woo-Yang Kim, Peng Jiang

DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

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Abstract

Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite–induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management.

Authors

Aida Habibovic, Milena Hristova, David E. Heppner, Karamatullah Danyal, Jennifer L. Ather, Yvonne M.W. Janssen-Heininger, Charles G. Irvin, Matthew E. Poynter, Lennart K. Lundblad, Anne E. Dixon, Miklos Geiszt, Albert van der Vliet