Bhallamudi, Sangeeta

AbstractAirway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain‐derived neurotrophic factor (BDNF). The glial cell‐derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+]i. Serum‐deprived ASM cells from non‐asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura‐2 loaded cells, acute GDNF or NRTN alone induced [Ca2+]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1‐Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1‐Fc chelator markedly decreased [Ca2+]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine‐induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Purpose Asthma prevalence has been shown higher in female as compared to male, strongly indicating the involvement of sex steroids in modulating airway hyperreactivity. Airway smooth muscle (ASM) cells are major structural cells of the airway that determine the airway tone. Our recent study demonstrated the presence of estrogen receptors (ERα and ERβ) in ASM cells and that both ER's are significantly upregulated during asthma and/or inflammation with pronounced ERβ expression. We previously reported that estrogen acutely reduces agonist‐induced intracellular calcium ([Ca 2+ ] i ) levels in ASM cells. However, the role of long‐term ER signaling in regulating [Ca 2+ ] i of ASM is unknown, especially during inflammation or in asthma. Accordingly, we hypothesized that estrogen receptor isoforms have differential roles in [Ca 2+ ] i regulation in ASM cells, with enhanced functionality during inflammation. Methods Human ASM cells were enzymatically dissociated from tissues obtained incidental to lung surgery (Mayo Clinic). Asthmatic and non‐asthmatic ASM cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence or absence of 17β‐estradiol (E 2 ), various ERα (PPT, THC) or ERβ (WAY, FERB, DPN) agonists and antagonists (MPP,PHTPP) for 24h. Cells were incubated with calcium indicator dye Fura‐2 AM and [Ca 2+ ] i responses in these cells were measured using various bronchoconstrictors such as bradykinin (BK,10nM), acetylcholine (Ach,1μM), histamine (His,10μM) and KCl (100μM) under Olympus epifluorescence imaging system. Results In both asthmatic and non‐asthmatic human ASM cells, ERβ agonists reduced [Ca 2+ ] i response to BK, His, Ach as compared to vehicle. Conversely E 2 and ERα agonists showed no change or a slight increase in [Ca 2+ ] i response compared to vehicle. The effectiveness of ERβ in reducing the [Ca 2+ ] i in human ASM cells was further confirmed by ERβ siRNA and ERβ overexpression studies. In the presence of inflammation induced by TNFα or IL‐13, ERβ agonists significantly reduced [Ca 2+ ] i response, while E 2 and ERα agonists did not draw out any notable changes. To understand the mechanistic basis of the ER specific signaling in [Ca 2+ ] i regulation, we measured L‐type calcium channel activity (using L‐type channel blocker nifedipine, 1μM) with KCl. ERβ agonist reduced KCl induced [Ca 2+ ] i increase, which was comparable to the effect of nifedipine. [Ca 2+ ] i reuptake pump (SERCA) which sequesters the cytosolic calcium into sarcoplasmic reticulum showed increased expression and activity in ASM cells with ERβ activation during inflammation. Conclusion Overall, our data highlights the presence of divergent ER signaling in [Ca 2+ ] i handling in ASM. Specifically ERβ activation is more effective in reducing [Ca 2+ ] i during inflammation, through both L‐type channel inhibition and increased SERCA activity. These varied signaling mechanisms could play a crucial role in smooth muscle contractility and tone in asthma. Support or Funding Information Supported by NIH grants R01 HL123494 (Venkatachalem), and R01 HL088029 (Prakash). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background The pathophysiology of asthma involves inflammation and altered structure and function of airway smooth muscle (ASM). There is emerging evidence that asthma affects adult women disproportionately with more severity than men. Also, our recent findings suggest that differential estrogen receptors (ER), ERα and ERβ expression and function are present in asthmatic ASM cells. Due to high oxygen environment in the lungs and the presence of inflammation, asthmatic ASM cells undergo oxidative stress. Mitochondrial dysfunction due to this oxidative stress plays a crucial role in the bioenergetics pathways involved in asthma. In this study, we hypothesize that differential ER signaling affects mitochondrial dynamics (fission/fusion) in ASM, which subsequently affects reactive oxygen species (ROS) generation and intracellular Ca 2+ ([Ca 2+ ] i ) buffering capacity of mitochondria. Methods Human ASM cells were enzymatically dissociated from lung samples of non‐asthmatics undergoing thoracic surgery at Mayo Clinic. Cells were grown onto 8‐well labtek plates and treated with pro‐inflammatory cytokine, TNFα (20 mg/mL) in the presence or absence of 1 nM 17β‐estradiol (E 2 ) which is a nonselective ER agonist, 10 nM PPT (ERα agonist) or 10 nM WAY (ERβ agonist) for 24h. For morphology studies, the cells were loaded with Mitotracker Green and then imaged on the Bio‐Tek Lionheart FX Live Cell Imager. Form factor and aspect ratio are used as parameters for determining fission vs fusion in mitochondria and these were calculated using MATLAB. Western blot and qPCR techniques were performed to access the protein and gene expression of the fission and fusion proteins Dynamin Related Protein1 (Drp1) and Mitofusion2 (Mfn2). ROS measurement was performed by loading cells with MitoSOX Red and their fluorescence intensity was recorded on the Live Cell Imager. Results In mitotracker loaded ASM cells, ERβ agonist WAY was effective in increasing the form factor and aspect ratio as compared to vehicle. In addition, ERβ was effective in reducing TNFα induced reduction in form factor and aspect ratio. However, both E 2 and ERα agonist, PPT, did not show any changes in form factor and aspect ratio of TNFα exposed ASM cells. Western analysis and gene expression studies showed higher expression of Drp1 and lower expression of Mfn2 in TNFα treated cells. Pre‐treatment with ERβ‐agonist reversed the altered expression of Drp1 and Mfn2 in TNFα treated cells. Using MitoSOX Red, it was observed that ERβ agonist WAY treated cells showed lower ROS generation as compared to TNFα. Conclusion Our data suggests a novel estrogen signaling in ASM cells for maintaining the structure of mitochondria. ERβ is effective in ameliorating the inflammation due to TNFα and hence preserves the morphology of mitochondria during inflammation. Further studies will confirm the subsequent alterations in mitochondrial ROS generation and [Ca 2+ ] i buffering capacity due to this altered mitochondrial morphology. Support or Funding Information Supported by the UGSRF fellowship by APS (Jesme) and NIH grants R01 HL123494, (Venkatachalem), and R01 HL 088029 (Prakash) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Purpose Bronchial asthma is a chronic inflammation of the airways that is characterized by airway remodeling, hyperactive responsiveness and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest that there is a higher incidence of asthma in pre‐menopausal women and aging men, strongly indicating the role of sex steroids in modulating the airway tone. A major sex steroid, estrogen mediates its role through estrogen receptors (ER) ‐ ERα and ERβ. Our recent study demonstrated that these receptors are expressed in both male and female ASM cells and their expression is upregulated in the presence of inflammation/asthma. However, the role of ER signaling in asthma or genomic mechanisms in intracellular Ca 2+ ([Ca 2+ ] i ) regulation in human ASM are unknown. In this study, we evaluated the genomic effect of specific ERα and ERβ modulators on the [Ca 2+ ] i in human ASM cells. Methods Human ASM cells were enzymatically dissociated from tissue obtained incidental to lung surgery from Mayo Clinic (IRB approved). Cells were plated onto 8‐well Lab‐Tek plates and grown to 70% confluence in a 5% CO 2 humidified incubator. Cells were exposed to pro‐inflammatory cytokines TNFα (20ng/ml) or IL‐13 (50ng/ml) in the presence and absence of 1nM 17β‐estradiol (E 2 ), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24h. Cells were washed with HBSS and incubated with 3 μm Fluo‐3 for 1h for calcium imaging. The [Ca 2+ ] i levels are measured from the intensity change observed following exposure to 10μM histamine under both physiological (2 mM) and zero extracellular calcium using Biotek LFX Fluorescence Imager. Results In fluo‐3 loaded ASM cells, 24h exposure to WAY (and to a lesser extent PPT and E 2 ) was effective in reducing [Ca 2+ ] i levels. TNFα and IL‐13 significantly increased [Ca 2+ ] i compared to vehicle. In the presence of TNFα or IL‐13, WAY significantly reduced [Ca 2+ ] i in ASM cells, while PPT did not draw out any notable changes compared to TNFα or IL‐13 treatment alone. Addition of nifedipine (1μM, L‐type calcium channel blocker) resulted in a ~40% decrease in [Ca 2+ ] i compared to control. This nifedipine blunting effect was similar in PPT treated group and to a higher extend to the WAY on [Ca 2+ ] i responses to histamine, suggesting that ER effects were mediated via L‐type calcium channels. To verify the ER effect on sarcoplasmic reticulum (SR) Ca 2+ reuptake, extracellular Ca 2+ was removed by perfusion with zero Ca 2+ HBSS, and Ca 2+ influx was non‐specifically blocked with 1 mM LaCl 3. Under these conditions, the [Ca 2+ ] i responses were significantly reduced in the presence of WAY. Further, we examined effects of ER signaling on [Ca 2+ ] i sequestration to SR (SERCA activity). In both control ASM cells as well as in those exposed to cytokines, WAY increased the rate of fall of [Ca 2+ ] i responses to histamine, suggesting greater sequestration of [Ca 2+ ] i. Conclusions Overall, our data highlights that both ERα and ERβ specific agonist were able to reduce [Ca 2+ ] i in the inflamed ASM via receptor‐mediated genomic action. Specifically, ERβ activation is more effective in the presence of inflammation. Support or Funding Information Supported by NIH grants R01 HL123494 and R01 HL123494‐02S1 (Venkatachalem) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Purpose Epidemiological evidences suggest higher incidence and severity of asthma in pre‐menopausal women and aging men, indicating the role of sex steroids, especially estrogen. Our recent studies demonstrated that airway smooth muscle (ASM) cells from both male and female expresses estrogen receptors (ERα and β) and these receptors differentially regulate intracellular calcium ([Ca 2+ ] i ) which is a key role player in contraction. The degree of force for contraction can also be altered in a Ca 2+ ‐independent manner via RhoA. Both Ca 2+ ‐dependent and Ca 2+ ‐independent pathways in ASM thus together contribute towards the airway contractility. In this study, we hypothesized that the ERs differentially regulate cyclic adenosine monophosphate (cAMP) and RhoA mechanisms, thereby affecting the overall contractility of human ASM cells. Methods Asthmatic and non‐asthmatic primary human ASM cells (isolated from lung tissue, Mayo Clinic, IRB‐approved) were seeded and grown to 70% confluence. After serum‐deprivation, cells were exposed to 1nM 17β‐estradiol (E 2 ), 10nM PPT (ERα agonist) or WAY (ERβ agonist) for 24hr. IBMX (10μM) was used as phosphodiesterase inhibitor and Isoproterol (100nM for 10min) served as a positive control group for estimation of cAMP. In parallel studies, phosphorylation of myosin light chain (MLC 20 ) and myosin phosphatase targeting subunit‐1 (MYPT1) was assessed through Western analyses. Activated RhoA and cAMP measurements were done through ELISA kits. Cell contraction was accessed through traction force microscopy (TFM) using softgel plates coated with collagen and fluorescent beads. Upon treatments, phase‐contrast and fluorescent images were captured before, during and after 10μM histamine exposure and net traction force was calculated using advanced software. Results Prolonged (24h) activation of ERβ significantly increased the baseline cAMP levels whereas ERα activation decreased it. This effect was more profound in asthmatic ASM cells compared to non‐asthmatics. Furthermore, ERβ activation lowered the active RhoA levels in non‐asthmatic human ASM cells. The phosphorylation of MLC 20 and MYPT1 was significantly attenuated with ERβ activation compared to vehicle. TFM studies demonstrated reduced traction force in non‐asthmatic ASM cells treated with ERβ agonist compared to the vehicle, whereas ERα activation did not show any significant changes. Conclusion Our findings highlight the crucial role of divergent ER signaling in regulating Ca 2+ ‐dependent and Ca 2+ ‐independent pathways, which together govern the contractile function of ASM. Particularly, ERβ activation (and not ERα) mediates ASM relaxation by increasing the cAMP, reducing active RhoA and decreasing phosphorylation of MLC 20 /MYPT1. Thus, ERβ activation might serve as a novel target to manage the increased ASM contractility in asthma. Support or Funding Information Acknowledgement Supported by NIH grants R01‐HL0123494 (Venkatachalem), R01‐HL 088029 (Prakash) and R01‐HL142061 (Pabelick, Prakash).

Asthma is defined as chronic inflammation of the airways and is characterized by airway remodeling, hyperresponsiveness, and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest a higher incidence and severity of asthma in adult women, indicating a concrete role of sex steroids in modulating the airway tone. Estrogen, a major female sex steroid mediates its role through estrogen receptors (ER) ERα and ERβ, which are shown to be expressed in human ASM, and their expression is upregulated in lung inflammation and asthma. Previous studies suggested rapid, nongenomic signaling of estrogen via ERs reduces intracellular calcium ([Ca2+]i), thereby promoting relaxation of ASM. However, long-term ER activation on [Ca2+]iregulation in human ASM during inflammation or in asthma is still not known. In Fura-2-loaded nonasthmatic and asthmatic human ASM cells, we found that prolonged (24 h) exposure to ERα agonist (PPT) increased [Ca2+]iresponse to histamine, whereas ERβ activation (WAY) led to decreased [Ca2+] compared with vehicle. This was further confirmed by ER overexpression and knockdown studies using various bronchoconstrictor agents. Interestingly, ERβ activation was more effective than 17β-estradiol in reducing [Ca2+]iresponses in the presence of TNF-α or IL-13, while no observable changes were noticed with PPT in the presence of either cytokine. The [Ca2+]i-reducing effects of ERβ were mediated partially via L-type calcium channel inhibition and increased Ca2+sequestration by sarcoplasmic reticulum.Overall, these data highlight the differential signaling of ERα and ERβ in ASM during inflammation. Specific ERβ activation reduces [Ca2+]iin the inflamed ASM cells and is likely to play a crucial role in regulating ASM contractility, thereby relaxing airways.