Epithelial Gpr116 regulates pulmonary alveolar homeostasis via G_q/11 signaling

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Abstract

Pulmonary function is dependent upon the precise regulation of alveolar surfactant. Alterations in pulmonary surfactant concentrations or function impair ventilation and cause tissue injury. Identification of the molecular pathways that sense and regulate endogenous alveolar surfactant concentrations, coupled with the ability to pharmacologically modulate them both positively and negatively, would be a major therapeutic advance for patients with acute and chronic lung diseases caused by disruption of surfactant homeostasis. The orphan adhesion GPCR GPR116 (also known as Adgrf5) is a critical regulator of alveolar surfactant concentrations. Here, we show that human and mouse GPR116 control surfactant secretion and reuptake in alveolar type II (AT2) cells by regulating guanine nucleotide–binding domain α q and 11 (Gq/11) signaling. Synthetic peptides derived from the ectodomain of GPR116 activated Gq/11-dependent inositol phosphate conversion, calcium mobilization, and cortical F-actin stabilization to inhibit surfactant secretion. AT2 cell–specific deletion of Gnaq and Gna11 phenocopied the accumulation of surfactant observed in Gpr116–/– mice. These data provide proof of concept that GPR116 is a plausible therapeutic target to modulate endogenous alveolar surfactant pools to treat pulmonary diseases associated with surfactant dysfunction.

Authors

Kari Brown, Alyssa Filuta, Marie-Gabrielle Ludwig, Klaus Seuwen, Julian Jaros, Solange Vidal, Kavisha Arora, Anjaparavanda P. Naren, Kathirvel Kandasamy, Kaushik Parthasarathi, Stefan Offermanns, Robert J. Mason, William E. Miller, Jeffrey A. Whitsett, James P. Bridges

Role of adenylyl cyclase 6 in the development of lithium-induced nephrogenic diabetes insipidus

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Abstract

Psychiatric patients treated with lithium (Li⁺) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li⁺-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li⁺ inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6^–/–) mice and potentially novel connecting tubule/principal cell–specific AC6 knockout (AC6^loxloxCre) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li⁺ administration increased water intake and reduced urine osmolality in control, AC6^–/–, and AC6^loxloxCre mice. Consistent with AC6^–/– mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6^loxloxCre mice compared with controls under standard conditions, and levels were further reduced after Li⁺ administration. AC6^loxloxCre and control mice had a similar increase in the numbers of proliferating cell nuclear antigen–positive cells in response to Li⁺. However, AC6^loxloxCre mice had a higher number of H⁺-ATPase B1 subunit–positive cells under standard conditions and after Li⁺ administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell–to–principal cell ratio.

Authors

Søren Brandt Poulsen, Tina Bøgelund Kristensen, Heddwen L. Brooks, Donald E. Kohan, Timo Rieg, Robert A. Fenton

Store-operated Ca²⁺ entry controls ameloblast cell function and enamel development

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Abstract

Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca²⁺ release–activated Ca²⁺ (CRAC) channels and store-operated Ca²⁺ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2^K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2^K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2^K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel.

Authors

Miriam Eckstein, Martin Vaeth, Cinzia Fornai, Manikandan Vinu, Timothy G. Bromage, Meerim K. Nurbaeva, Jessica L. Sorge, Paulo G. Coelho, Youssef Idaghdour, Stefan Feske, Rodrigo S. Lacruz

Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

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Abstract

Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KL_Lenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KL_Lenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

Authors

Melissa Gilbert-Ross, Jessica Konen, Junghui Koo, John Shupe, Brian S. Robinson, Walter Guy Wiles IV, Chunzi Huang, W. David Martin, Madhusmita Behera, Geoffrey H. Smith, Charles E. Hill, Michael R. Rossi, Gabriel L. Sica, Manali Rupji, Zhengjia Chen, Jeanne Kowalski, Andrea L. Kasinski, Suresh S. Ramalingam, Haian Fu, Fadlo R. Khuri, Wei Zhou, Adam I. Marcus

The volume-regulated anion channel (LRRC8) in nodose neurons is sensitive to acidic pH

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Abstract

The leucine rich repeat containing protein 8A (LRRC8A), or SWELL1, is an essential component of the volume-regulated anion channel (VRAC) that is activated by cell swelling and ionic strength. We report here for the first time to our knowledge its expression in a primary cell culture of nodose ganglia neurons and its localization in the soma, neurites, and neuronal membrane. We show that this neuronal VRAC/SWELL1 senses low external pH (pH_o) in addition to hypoosmolarity. A robust sustained chloride current is seen in 77% of isolated nodose neurons following brief exposures to extracellular acid pH. Its activation involves proton efflux, intracellular alkalinity, and an increase in NOX-derived H₂O_2. The molecular identity of both the hypoosmolarity-induced and acid pH_o–conditioned VRAC as LRRC8A (SWELL1) was confirmed by Cre-flox–mediated KO, shRNA-mediated knockdown, and CRISPR/Cas9-mediated LRRC8A deletion in HEK cells and in primary nodose neuronal cultures. Activation of VRAC by low pH_o reduces neuronal injury during simulated ischemia and N-methyl-D-aspartate–induced (NMDA-induced) apoptosis. These results identify the VRAC (LRRC8A) as a dual sensor of hypoosmolarity and low pH_o in vagal afferent neurons and define the mechanisms of its activation and its neuroprotective potential.

Authors

Runping Wang, Yongjun Lu, Susheel Gunasekar, Yanhui Zhang, Christopher J. Benson, Mark W. Chapleau, Rajan Sah, François M. Abboud

Muscular dystrophy in PTFR/cavin-1 null mice

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Abstract

ice and humans lacking the caveolae component polymerase I transcription release factor (PTRF, also known as cavin-1) exhibit lipo- and muscular dystrophy. Here we describe the molecular features underlying the muscle phenotype for PTRF/cavin-1 null mice. These animals had a decreased ability to exercise, and exhibited muscle hypertrophy with increased muscle fiber size and muscle mass due, in part, to constitutive activation of the Akt pathway. Their muscles were fibrotic and exhibited impaired membrane integrity accompanied by an apparent compensatory activation of the dystrophin-glycoprotein complex along with elevated expression of proteins involved in muscle repair function. Ptrf deletion also caused decreased mitochondrial function, oxygen consumption, and altered myofiber composition. Thus, in addition to compromised adipocyte-related physiology, the absence of PTRF/cavin-1 in mice caused a unique form of muscular dystrophy with a phenotype similar or identical to that seen in humans lacking this protein. Further understanding of this muscular dystrophy model will provide information relevant to the human situation and guidance for potential therapies.

Authors

Shi-Ying Ding, Libin Liu, Paul F. Pilch

Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass

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Abstract

Mitophagy occurs during ischemia/reperfusion (I/R) and limits oxidative stress and injury. Mitochondrial turnover was assessed in patients undergoing cardiac surgery involving cardiopulmonary bypass (CPB). Paired biopsies of right atrial appendage before initiation and after weaning from CPB were processed for protein analysis, mitochondrial DNA/nuclear DNA ratio (mtDNA:nucDNA ratio), mtDNA damage, mRNA, and polysome profiling. Mitophagy in the post-CPB samples was evidenced by decreased levels of mitophagy adapters NDP52 and optineurin in whole tissue lysate, decreased Opa1 long form, and translocation of Parkin to the mitochondrial fraction. PCR analysis of mtDNA comparing amplification of short vs. long segments of mtDNA revealed increased damage following cardiac surgery. Surprisingly, a marked increase in several mitochondria-specific protein markers and mtDNA:nucDNA ratio was observed, consistent with increased mitochondrial biogenesis. mRNA analysis suggested that mitochondrial biogenesis was traniscription independent and likely driven by increased translation of existing mRNAs. These findings demonstrate in humans that both mitophagy and mitochondrial biogenesis occur during cardiac surgery involving CPB. We suggest that mitophagy is balanced by mitochondrial biogenesis during I/R stress experienced during surgery. Mitigating mtDNA damage and elucidating mechanisms regulating mitochondrial turnover will lead to interventions to improve outcome after I/R in the setting of heart disease.

Authors

Allen M. Andres, Kyle C. Tucker, Amandine Thomas, David J.R. Taylor, David Sengstock, Salik M. Jahania, Reza Dabir, Somayeh Pourpirali, Jamelle A. Brown, David G. Westbrook, Scott W. Ballinger, Robert M. Mentzer Jr., Roberta A. Gottlieb

Polyfunctional and IFN-γ monofunctional human CD4⁺ T cell populations are molecularly distinct

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Abstract

Pathogen-specific polyfunctional T cell responses have been associated with favorable clinical outcomes, but it is not known whether molecular differences exist between polyfunctional and monofunctional cytokine-producing T cells. Here, we report that polyfunctional CD4⁺ T cells induced during Plasmodium falciparum (P. falciparum) blood-stage infection in humans have a unique transcriptomic profile compared with IFN-γ monofunctional CD4⁺ T cells and, thus, are molecularly distinct. The 14-gene signature revealed in P. falciparum–reactive polyfunctional T cells is associated with cytokine signaling and lymphocyte chemotaxis, and systems biology analysis identified IL-27 as an upstream regulator of the polyfunctional gene signature. Importantly, the polyfunctional gene signature is largely conserved in Influenza-reactive polyfunctional CD4⁺ T cells, suggesting that polyfunctional T cells have core characteristics independent of pathogen specificity. This study provides the first evidence to our knowledge that consistent molecular differences exist between polyfunctional and monofunctional CD4⁺ T cells.

Authors

Julie G. Burel, Simon H. Apte, Penny L. Groves, James S. McCarthy, Denise L. Doolan

Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension

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Abstract

Amphetamine (AMPH) or methamphetamine (METH) abuse can cause oxidative damage and is a risk factor for diseases including pulmonary arterial hypertension (PAH). Pulmonary artery endothelial cells (PAECs) from AMPH-associated-PAH patients show DNA damage as judged by γH2AX foci and DNA comet tails. We therefore hypothesized that AMPH induces DNA damage and vascular pathology by interfering with normal adaptation to an environmental perturbation causing oxidative stress. Consistent with this, we found that AMPH alone does not cause DNA damage in normoxic PAECs, but greatly amplifies DNA damage in hypoxic PAECs. The mechanism involves AMPH activation of protein phosphatase 2A, which potentiates inhibition of Akt. This increases sirtuin 1, causing deacetylation and degradation of HIF1α, thereby impairing its transcriptional activity, resulting in a reduction in pyruvate dehydrogenase kinase 1 and impaired cytochrome c oxidase 4 isoform switch. Mitochondrial oxidative phosphorylation is inappropriately enhanced and, as a result of impaired electron transport and mitochondrial ROS increase, caspase-3 is activated and DNA damage is induced. In mice given binge doses of METH followed by hypoxia, HIF1α is suppressed and pulmonary artery DNA damage foci are associated with worse pulmonary vascular remodeling. Thus, chronic AMPH/METH can induce DNA damage associated with vascular disease by subverting the adaptive responses to oxidative stress.

Authors

Pin-I Chen, Aiqin Cao, Kazuya Miyagawa, Nancy F. Tojais, Jan K. Hennigs, Caiyun G. Li, Nathaly M. Sweeney, Audrey S. Inglis, Lingli Wang, Dan Li, Matthew Ye, Brian J. Feldman, Marlene Rabinovitch

Vps34 regulates myofibril proteostasis to prevent hypertrophic cardiomyopathy

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Abstract

Hypertrophic cardiomyopathy (HCM) is a common heart disease with a prevalence of 1 in 500 in the general population. Several mutations in genes encoding cardiac proteins have been found in HCM patients, but these changes do not predict occurrence or prognosis and the molecular mechanisms underlying HCM remain largely elusive. Here we show that cardiac expression of vacuolar protein sorting 34 (Vps34) is reduced in a subset of HCM patients. In a mouse model, muscle-specific loss of Vps34 led to HCM-like manifestations and sudden death. Vps34-deficient hearts exhibited abnormal histopathologies, including myofibrillar disarray and aggregates containing αB-crystallin (CryAB). These features result from a block in the ESCRT-mediated proteolysis that normally degrades K63-polyubiquitinated CryAB. CryAB deposition was also found in myocardial specimens from a subset of HCM patients whose hearts showed decreased Vps34. Our results identify disruption of the previously unknown Vps34-CryAB axis as a potentially novel etiology of HCM.

Authors

Hirotaka Kimura, Satoshi Eguchi, Junko Sasaki, Keiji Kuba, Hiroki Nakanishi, Shunsuke Takasuga, Masakazu Yamazaki, Akiteru Goto, Hiroyuki Watanabe, Hiroshi Itoh, Yumiko Imai, Akira Suzuki, Noboru Mizushima, Takehiko Sasaki