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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: J Cell Physiol. 2021 Jul 18;237(1):603–616. doi: 10.1002/jcp.30528

Autocrine regulation of airway smooth muscle contraction by diacylglycerol kinase

Santosh K Yadav 1, Pawan Sharma 1, Sushrut D Shah 1, Reynold A Panettieri Jr 2, Taku Kambayashi 3, Raymond B Penn 1, Deepak A Deshpande 1,*
PMCID: PMC8763953  NIHMSID: NIHMS1722525  PMID: 34278583

Abstract

Diacylglycerol kinase (DGK), a lipid kinase, catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid, thereby terminating DAG-mediated signaling by Gq-coupled receptors that regulate contraction of airway smooth muscle (ASM). A previous study from our laboratory demonstrated that DGK inhibition or genetic ablation leads to reduced ASM contraction and provides protection for allergen-induced airway hyperresponsiveness. However, the mechanism by which DGK regulates contractile signaling in ASM is not well established. Herein, we investigated the role of pro-relaxant cAMP-protein kinase A (PKA) signaling in DGK-mediated regulation of ASM contraction. Pretreatment of human ASM cells with DGK inhibitor I activated PKA as demonstrated by the phosphorylation of PKA substrates, VASP, Hsp20, and CREB, which was abrogated when PKA was inhibited pharmacologically or molecularly using overexpression of PKA inhibitor peptide, PKI. Further, inhibition of DGK resulted in induction of cyclooxygenase (COX) and generation of prostaglandin E2 (PGE2) with concomitant activation of Gs-cAMP-PKA signaling in ASM cells in an autocrine/paracrine fashion. Inhibition of protein kinase C (PKC) or extracellular-signal-regulated kinase (ERK) attenuated DGK-mediated production of PGE2 and activation of cAMP-PKA signaling in human ASM cells, suggesting that inhibition of DGK activates the COX-PGE2 pathway in a PKC-ERK-dependent manner. Finally, DGK inhibition-mediated attenuation of contractile agonist-induced phosphorylation of myosin light chain (MLC)-20, a marker of ASM contraction, involves COX-mediated cAMP production and PKA activation in ASM cells. Collectively these findings establish a novel mechanism by which DGK regulates ASM contraction and further advances DGK as a potential therapeutic target to provide effective bronchoprotection in asthma.

Keywords: cyclooxygenase, smooth muscle, GPCR, calcium, DGK, asthma

Introduction

Excessive airway smooth muscle contraction (ASM) is a key feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (Bowers & Dahm, 1993). ASM contraction is predominantly regulated by G protein-coupled receptors (GPCRs) expressed on the sarcolemma of ASM cells. Endogenous agonists from parasympathetic nerve terminals (e.g., acetylcholine) as well as inflammatory mediators (e.g., histamine, leukotrienes) exert pro-contractile actions on ASM by activating signaling through the Gq class of GPCRs (Billington & Penn, 2003). Therefore, delineating mechanisms by which ASM contraction is regulated provides unique opportunities to develop novel therapies to protect against bronchoconstriction.

Gq activation in ASM cells stimulates activation of phospholipase C (PLC), which converts phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG) (Penn & Benovic, 2008; Sternweis & Smrcka, 1992). IP3 triggers calcium (Ca2+) release from the intracellular stores, which is the first critical step in initiating contractile signaling. In addition, DAG activates PKC and promotes contraction via activation of multiple signaling molecules, including Rho and Rho kinase (Rhee & Bae, 1997). While IP3-Ca2+-mediated contractile signaling is terminated by multiple mechanisms of Ca2+ reuptake in or extrusion from the intracellular stores of ASM cells or extracellular space (Hirota, Helli, & Janssen, 2007), the DAG-mediated signaling is terminated by conversion of DAG into phosphatidic acid (PA) catalyzed by diacylglycerol kinase (DGK) enzyme. DGKs are ubiquitously expressed and are classified into five different groups namely, Type I (α, β, γ), II (δ, η, κ), III (ε), IV (ζ, ι) and V (θ ) (Topham & Epand, 2009; van Blitterswijk & Houssa, 2000). Our recent study demonstrated genetic ablation (α and ζ isoforms) and pharmacological targeting of DGK is protective in allergic airway inflammation and ASM hyper-contraction (Singh et al., 2019). However, the mechanism by which DGK regulates ASM contraction is not fully established.

Gs-coupled GPCRs mediate Pro-relaxant signaling that counterbalances Gq signaling in ASM cells. Activation by Gs-coupled GPCR by various endogenous/exogenous agonists (e.g., beta-agonists, prostaglandin E2) results in the activation of heterotrimeric Gs and dissociation of its α and βγ subunits (Billington & Penn, 2003). The α subunit directly interacts with and activates adenylyl cyclase, which hydrolyzes ATP to generate cyclic adenosine monophosphate (cAMP). cAMP inhibits ASM contraction via activation of the cAMP-dependent protein kinase, aka protein kinase A (PKA) (Billington & Penn, 2003). Epinephrine, an endogenous Gs-coupled GPCR agonist, is released from autonomic nerve terminals and PGE2 produced by cyclooxygenase (COX). Studies, including ours, have shown that a fluctuation in the levels of COX-2 activity and endogenous PGE2 underpin changes in ASM reactivity, ASM proliferation, and airway inflammation in experimental models of asthma (Gavett et al., 1999; Guo et al., 2005; Misior et al., 2008; Pascual et al., 2001; Pascual et al., 2006; Peebles et al., 2002; Torres et al., 2008; Yan et al., 2011). Additionally, it has been reported that DGKε plays a role in the induction of COX in other organs (Lukiw, Cui, Musto, Musto, & Bazan, 2005; Nakano, Topham, & Goto, 2020; Zhu et al., 2016). However, it is not yet clear if DGK isoforms modulate COX activation in the lung.

Herein, we sought to investigate the role of cAMP-PKA in DGK-mediated regulation of ASM contraction. We tested the effect of inhibition of DGK on PKA activation and established the mechanism by which DGK inhibition promotes PKA activation in human ASM cells. Inhibition of DGK increases prostaglandin E2 (PGE2) levels via a PKC-ERK-dependent induction of cyclooxygenase (COX) that increases PGE2 levels to promote cAMP-PKA signaling and inhibit ASM contraction. These findings establish a novel mechanism by which DGK regulates ASM contraction and further advances DGK as a potential therapeutic target to provide effective bronchoprotection in asthma.

Materials and Methods

Materials

The primary antibody against total VASP (610448) was from BD biosciences (Franklin Lakes, NJ, USA). Anti-pCREB (9198S) and RIPA cell lysis buffer (9806) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-pHsp20 (58522) and anti-β-actin (58522) were purchased from Abcam (Cambridge MA, USA) and Sigma (St. Louis, MO, USA) respectively. Secondary antibodies IRDye 680RD or 800CW were from LI-COR (Lincoln, NE, USA). DGK inhibitor I (R59022), 3-Isobutyl-1-methylxanthine (IBMX), forskolin, histamine, H-89 dihydrochloride hydrate, indomethacin, and all other standard reagents were obtained from Sigma Life Sciences (St. Louis, MO, USA) unless otherwise mentioned. Protease and phosphatase inhibitors were from Bimake (Houston, TX, USA). Bisindolylmaleimide I (13298) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). U0126 (S1102) was procured from Selleckchem (Houston, TX, USA). All polyacrylamide gel casting, running, and transfer reagents and equipment were from Bio-Rad Laboratories (Hercules, CA, USA) or from previously identified sources (Michael et al., 2019; Sharma et al., 2016).

Cell culture

Human ASM cells were isolated from different lung donors, and cultured using complete F-12 media supplemented with 10% FBS, penicillin/streptomycin, HEPES buffer, CaCl2, L-Glutamine (Gibco), and NaOH between 2-6 passages (Panettieri, Murray, DePalo, Yadvish, & Kotlikoff, 1989). Human ASM cell cultures were replenished with fresh media every alternate day. For western blotting, cAMP, and PGE2 assay, cells were seeded in 12-well plates, and for CRE-Luciferase (CRE-Luc) assay, cells were seeded in 96-well plates. Cells were cultured till full confluency and then growth arrested with F-12 medium containing 1% insulin-transferrin without serum for 24 or 48 h. In a select set of experiments, cells were pretreated with different inhibitors: DGKI (DGK inhibitor I, 30 μM) (Singh et al., 2019), H-89 dihydrochloride (H-89, PKA inhibitor, 10 μM) (Taylor, Pantazaka, Shelley, & Taylor, 2017), indomethacin (pan COX inhibitor, 10 μM) (Misior et al., 2008), SC560 (COX-1 inhibitor, 10 nM) (S. Ito et al., 2006), NS398 (COX-2 inhibitor, 1 μM) (Baarschers & Pachler, 1965), bisindolylmaleimide I (Bis-I, PKC inhibitor, 10 μM) (Misior et al., 2008), U0126 (MEK inhibitor, 1 μM) (Defnet et al., 2019), or isobutyl methylxanthine (phosphodiesterase inhibitor (IBMX), 1mM) (Michael et al., 2019) for the indicated time. The concentration of various pharmacological inhibitors used in this study was based on our previous publications or as reported by other researchers.

Viral transfection of human ASM cells

Retrovirus for the expression of green fluorescent protein (GFP) or the GFP-chimera of the PKA inhibitory peptide (PKI) was produced by cotransfecting GP2-293 cells with p-vesicular stomatitis virus (VSV)-G vector (encoding the pantropic VSV-G envelope protein) and either pLPCX-GFP or pLPCX-PKI-GFP. Culture media containing viral particles was harvested after 48 h of transfection and used to infect primary human ASM cell cultures as previously described (Guo et al., 2005; Michael et al., 2019; Morgan et al., 2014). Human ASM cell cultures were subsequently selected for homogeneity with 5 μg/ml puromycin (Tocris Bioscience, UK).

Human ASM cells were transfected with lentiviral particles encoding luciferase reporter under the control of the cAMP response element (CRE) promoter (Qiagen, Germantown, MA, USA). Cignal Lenti Reporter Assays utilize a unique combination of multiple repeats of a CRE transcription factor binding site and essential promoter elements to drive the expression of a reporter gene (firefly luciferase) coupled with lentiviral delivery as described previously (Yan et al., 2011). Cells were used for experiments 24 h after transfection as described below.

Western blotting

Human ASM cells were lysed in RIPA buffer (CST, USA) supplemented with protease and phosphatase inhibitor (Bimake) at 4°C for 30 min. Lysates were then mixed with Laemmli buffer (Bio-Rad) containing 10% β-mercaptoethanol. Protein samples were boiled at 95°C for 5 min, separated on SDS-PAGE, and transferred onto a nitrocellulose membrane. Target proteins were detected via incubation with respective primary antibodies overnight in TBST with 3% BSA. A secondary antibody conjugated with an infrared dye either at 600 nm or 800 nm wavelength was used to detect target protein using Odyssey infrared scanner (LI-COR Biosciences, Lincoln, USA). Protein band intensity was quantified using Odyssey software as described previously (Deshpande et al., 2010).

Protein samples for pMLC immunoblot analysis were prepared by treating the cells with stop buffer (water: perchloric acid in 1:1 ratio) for 5 min on ice and then pelleting by centrifugation. The cell pellet was then washed two times in ice-cold water to remove excess perchloric acid, followed by lysis in RIPA buffer and denaturation in LDS buffer followed by boiling at 70°C for 10 min. Samples were run on precast gels (Novex) and transferred onto a nitrocellulose membrane using mini iBlot (Invitrogen). Proteins on the membrane were probed using anti-pMLC (CST#3674) or anti-β-actin (Sigma) antibodies followed by detection as described above and plotted as a fold change to vehicle-treated samples after normalizing the values to β-actin band intensity.

cAMP assay

Human ASM cells were grown to full confluency in 12-well plates, serum-starved for 24 h, and then incubated with 1 mM IBMX for 30 min. Cells were then incubated with 30 μM DGK inhibitor I for 15 min followed by stimulation with 100 μM forskolin, or 1 μM isoproterenol for 15 min. In select experiments, cells were treated with indomethacin, Bis-I, or U0126 for 20 min followed by 30 μM DGK inhibitor I for 15 min. and subsequently stimulated with 100 μM forskolin, or 1 μM isoproterenol for 15 min. After treatment, cells were lysed in cAMP lysis buffer, and cAMP levels were measured by ELISA (#4412183, Fisher Scientific, USA) as described previously (Kong et al., 2008) per the manufacturer's protocol.

PGE2 assay

Human ASM cells were seeded in 12-well plates, and after 24 h, cells were growth-arrested in F-12 media containing 1% insulin and transferrin. Cells were incubated with different inhibitors (indomethacin, Bis-I, or U0126) for 20 min followed by 30 μM DGK inhibitor I for 15 min in F-12 media. The supernatant was collected at different time points to determine the concentration of PGE2 using a competitive ELISA approach (# 514010, Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer's protocol.

CRE-Luc assay

Human ASM cells were transiently transfected with CRE-Luc lentivirus (Qiagen, Germantown, MA, USA) in a 10 cm dish as described previously (Yan et al., 2011). Twenty-four hours post-transfection, cells were trypsinized and reseeded in 96-well plates at a seeding density of 10,000 cells per well. Cells were serum-starved in growth arresting media for 24 h and incubated with H-89, indomethacin, SC560, NS398, Bis-I, or U0126 for 20 min followed by stimulation with 30 μM DGK inhibitor I for 6 h. Cells were lysed, and luciferase luminescence was recorded following the manufacturer's protocol (Applied Biosystems) using a FlexStation III plate reader.

Statistical Analysis

All data are presented as mean ± SEM values from the 'n' number of lines derived from different donors. Densitometry data of western blot are normalized using band intensities in vehicle-treated cells. VASP phosphorylation data were analyzed by calculating the phosphorylated VASP as a percentage of total VASP under different experimental conditions. cAMP and PGE2 concentrations were determined by extrapolation from a standard curve. One-way ANOVA with Bonferroni post hoc analysis or Student's t-test was used to determine statistical differences among treatment groups using GraphPad Prism VI software (La Jolla, CA, USA). A p ≤ 0.05 was considered sufficient to reject the null hypothesis.

Results

Effect of DGK inhibition on PKA activation

To determine the effect of DGK inhibition on PKA activity, we assessed phosphorylation of PKA substrates vasodilator-stimulated phosphoprotein (VASP), heat shock protein 20 (Hsp20), and cAMP response element-binding protein (CREB). Treatment of human ASM cells with DGK inhibitor I (30 μM, 15 min) resulted in a significant (p<0.05, n=5) increase in phosphorylation of VASP, CREB, and Hsp20 compared to vehicle (DMSO)-treated cells (Fig. 1). Further, pretreatment with PKA inhibitor H-89 (10 μM for 20 min) inhibited DGK inhibitor I-induced phosphorylation of VASP, CREB, and Hsp20 (Fig. 1 A-D; p<0.05). Human ASM cells treated with 1 μM isoproterenol for 15 min were used as a positive control in these experiments (Fig. 1 A-D). PKA-mediated activation of the cyclic AMP response element (CRE) was assessed using a luciferase reporter assay. Human ASM cells were treated with vehicle or H-89 (10 μM for 20 min) followed by treatment with 30 μM DGK inhibitor I for 6 h. DGK inhibition in human ASM cells significantly (p<0.05, n=5) increased CRE-Luc activity, which was attenuated by pretreatment with H-89 (Fig. 1 E; p<0.05).

Figure 1: Effect of DGK inhibition on PKA activation.

Figure 1:

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Human ASM cells were treated with 30 μM DGK inhibitor or 1 μM Iso (PKA activator) preincubated with 10 μM H-89 (PKA inhibitor) cells were lysed, and proteins were separated on SDS page and blotted for total VASP, pCREB, pHsp20 and βactin (A) Graphical representation of mean ± SEM of densitometry data of pVASP (B) pCREB (C), and pHsp20 (D) densitometry data (presented as % of total VASP for pVASP, and fold change to vehicle-treated cells for pCREB and pHsp20). Human ASM cells stably expressing CRE-Luc were stimulated with 10 μM H-89 (PKA inhibitor) for 20 min followed by 30 μM DGK inhibitor I for 6 h. Luminesce was recorded and represented as fold change to vehicle-treated cells (E). Data above is mean ± SEM of (n=5) independent experiments; *p<0.05 using one-way-ANOVA with Bonferroni post-hoc analysis. B: Basal; Iso: Isoproterenol.

We further validated our pharmacological studies using ASM cells stably expressing PKA inhibitory peptide PKI. Human ASM cells expressing GFP or GFP-PKI were treated with 30 μM DGK inhibitor I or 1 μM isoproterenol for 15 min, and phosphorylation of VASP and Hsp20 was assessed in cell lysates (Fig. 2 A-C). DGK inhibition resulted in increased phosphorylation of VASP and Hsp20 compared to vehicle-treatment in GFP expressing cells, but not in GFP-PKI expressing cells (Fig. 2 B and C; p<0.05). Further, PKA-mediated CRE-Luc activity was determined using human ASM cells stably expressing GFP and GFP-PKI. The CRE-Luc activity was enhanced by treatment with DGK inhibitor I (30 μM, 6 h) in GFP cells, but not in GFP-PKI cells, compared to treatment with DMSO (Fig. 2 D; p<0.05). Findings from these studies indicate that DGK inhibition in human ASM cells leads to the activation of PKA.

Figure 2: Effect of DGK inhibition on PKA activation.

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PKI-GFP and GFP stable human ASM cells were treated with 30 μM DGK inhibitor I or 1 μM Iso. The whole-cell lysate was obtained as described in the methods and was subjected to western blotting using anti-β-actin, total VASP, pHsp20, and GFP antibody (A). Graphical representation of mean ± SEM of the pVASP (B), and pHsp20 (C) densitometry data (presented as % of total VASP for pVASP, and fold change to vehicle-treated cells for pHsp20). PKI-GFP and GFP stable human ASM cells were transiently transfected with CRE-Luc construct and subjected to 30 μM DGK inhibitor I treatment for 6 h. Cells were lysed, and luminescence was recorded and plotted as fold change to luminescence detected in vehicle-treated cells (D). Data above is mean ± SEM of (n=4) independent experiments; *p<0.05 using one-way-ANOVA with Bonferroni post-hoc analysis. Veh: Vehicle; Iso: Isoproterenol.

Role of cyclooxygenase (COX) enzyme in DGK-mediated PKA activation

To establish the mechanism by which DGK regulates PKA activation, we hypothesized that DGK inhibition leads to the activation of cyclooxygenase. Human ASM cells were pre-incubated with SC560 (COX-1 inhibitor; 10 nM, 20 min), NS398 (COX-2 inhibitor; 1 μM, 20 min), or indomethacin (pan COX inhibitor; 10 μM, 20 min) followed by vehicle or DGK inhibitor I (30 μM, 15 min) and phosphorylation of VASP, and Hsp20 was assessed (Fig. 3 A-C). Treatment with bradykinin (1 μM, 15 min) was used as a positive control for COX activation. DGK inhibition resulted in phosphorylation of VASP and Hsp20, which were significantly attenuated by indomethacin and NS398 (Fig. 3 B, and C; p<0.05).

Figure 3: Effect of DGK inhibition on COX-mediated PKA activation.

Figure 3:

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Human ASM cells were pretreated with 10 nM SC560 (COX-1 inhibitor), 1 μM NS398 (COX-2 inhibitor), and 10 μM Indomethacin (pan COX inhibitor) for 20 min followed by stimulation with 30 μM DGK inhibitor I or 1 μM bradykinin (COX inducer) for 15 min. Cells were lysed, and immunoblot was performed for assessing total VASP and pHsp20 (A) levels. Graphical representation of mean ± SEM of the pVASP (B) and pHsp20 (C) densitometry data (presented as % of total VASP for pVASP, and fold change to vehicle-treated cells for pHsp20). Human ASM cells were pre-incubated with 10 μM Indo (pan COX inhibitor) for 20 min followed by stimulation with 30 μM DGK inhibitor I or 1 μM bradykinin (COX inducer) for 15 min, and levels of PGE2 (D) and intracellular cAMP (E) were measured. Human ASM cells stably expressing CRE-Luc were pre-incubated with 10 nM SC560 (COX-1 inhibitor), 1 μM NS398 (COX-2 inhibitor), and 10 μM Indo (pan COX inhibitor) for 20 min followed by stimulation with 30 μM DGK inhibitor I for 6 h. Cells were lysed, and luminescence was recorded and plotted as fold change to luminescence detected in vehicle-treated cells (F). Data above is mean ± SEM of (n=4) independent experiments; *p<0.05 using one-way-ANOVA with Bonferroni post-hoc analysis. Veh: Vehicle; Bk: Bradykinin; Indo: Indomethacin.

Activation of the COX enzyme is expected to result in the production of PGE2. Therefore, we measured PGE2 production in human ASM cells by ELISA. DGK inhibition significantly (n=5, p<0.05) increased PGE2 production, which was attenuated by pretreatment with indomethacin (Fig. 3D). In addition, DGK inhibition resulted in increased accumulation of cAMP in human ASM cells compared to vehicle-treated cells, which was significantly (n=5, p<0.05) attenuated by pretreatment with indomethacin (Fig. 3E). As expected, forskolin-induced cAMP elevation was not affected by treatment with indomethacin. Further, we tested the effect of COX inhibitors on DGK-mediated activation of CRE-Luc activity. DGK inhibition (30 μM, 6 h) significantly increased CRE-Luc activity compared to vehicle-treated cells, which was significantly attenuated by indomethacin and NS398 (Fig. 3F; p<0.05). These data collectively suggest that DGK inhibition leads to activation of COX enzyme and production of PGE2, which in turn activates the cAMP-PKA pathway in human ASM cells.

Role of protein kinase C (PKC) in DGK-mediated activation of PKA

PKC is the downstream effector of DAG and is a known activator of COX-mediated PGE2 synthesis (Pascual et al., 2006). We, therefore, investigated the role of PKC in DGK-mediated activation of PKA in human ASM cells. Human ASM cells were pretreated with Bis-I (PKC inhibitor; 10 μM, 20 min) followed by treatment with DGK inhibitor I (30 μM, 15 min) (Fig. 4 A-D), and phosphorylation of VASP, CREB, and Hsp20 was assessed by western blotting. There was a significant increase in phosphorylation of VASP, CREB, and Hsp20 by DGK inhibition compared to vehicle treatment, which was attenuated by Bis-I pretreatment (Fig. 4 B, C, D; p<0.05). Next, we assessed the effect of PKC inhibition on DGK-mediated PGE2 synthesis and cAMP generation in human ASM cells. DGK inhibition (30 μM) significantly increased PGE2 production and cAMP accumulation compared to vehicle-treated cells, which were significantly attenuated by PKC inhibition (Fig. 4 E, F p>0.05). Further, DGK inhibition (30 μM, 6 h)-induced activation of CRE-Luc activity was attenuated by PKC inhibitor Bis-I (Fig. 4 G; p<0.05).

Figure 4: Role of PKC in the activation of PKA by DGK inhibition.

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Human ASM cells were pretreated with 10 μM Bis-I (PKC inhibitor) for 20 min, followed by 30 μM DGK inhibitor I for 15 min. Cells were lysed and immunoblotted for total VASP, pCREB, and pHsp20 (A). Graphical representation of mean ± SEM of the pVASP (B), pCREB (C), and pHsp20 (D) (presented as % of total VASP for pVASP, and fold change to vehicle-treated cells for pCREB and pHsp20). Human ASM cells were pre-incubated with 10 μM Bis-I (PKC inhibitor) for 20 min followed by 30 μM DGK inhibitor I for 15 min, and levels of PGE2 (E) and intracellular cAMP (F) were measured using ELISA-based assays. Human ASM cells stably expressing CRE-Luc were stimulated with 30 μM DGK inhibitor I in the presence of vehicle or 10 μM Bis-I for 6 h. Cells were lysed, and luminescence was recorded and plotted as fold change to luminescence detected in vehicle-treated cells (G). Data above is mean ± SEM of n=5 independent experiments; *p<0.05 using one-way-ANOVA with Bonferroni post-hoc analysis. Veh: Vehicle; Bis-I: Bisindolylmaleimide I.

Role of MEK/ERK pathway in DGK-mediated PKA activation

Activation of COX signaling is known to be mediated by activation of MEK/ERK pathway (S. F. Luo, Wang, Chien, Hsiao, & Yang, 2003; C. M. Yang, Chien, Hsiao, Luo, & Wang, 2002). Therefore, we assessed the role of MEK/ERK in DGK inhibition-mediated PKA activation. Human ASM cells pretreated with U0126 (MEK inhibitor; 1 μM, 20 min) were treated with vehicle or DGK inhibitor I (30 μM, 15 min), and PKA activation was assessed by immunoblotting (Fig. 5 A-D). DGK inhibition increased phosphorylation of VASP, CREB, and Hsp20 compared to vehicle-treated cells, which was significantly attenuated by MEK inhibition (Fig. 5 B, C, D; p<0.05). Next, DGK inhibition significantly increased PGE2 production and cAMP accumulation compared to vehicle-treated cells, which was significantly attenuated by MEK inhibition (Fig. 5 E, F; p<0.05). Further, we assessed the effect of MEK inhibition on DGK-mediated activation of CRE-Luc activity. Treatment of human ASM cells with DGK inhibitor I (30 μM, 6 h) significantly increased CRE-Luc activity compared to vehicle-treated cells, which was significantly attenuated by U0126 (Fig. 5 G; p<0.05). These data collectively suggest that DGK-mediated induction of the COX enzyme involves activation of the MEK/ERK pathway.

Figure 5: Effect of DGK inhibition on MEK mediated PKA activation.

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Human ASM cells were pretreated with 1 μM U0126 (MEK inhibitor) for 20 min, followed by 30 μM DGK inhibitor I for 15 min. Cells were lysed and immunoblotted for total VASP, pCREB, and pHsp20 (A) levels. Graphical representation of mean ± SEM of the pVASP (B), pCREB (C), and pHsp20 (D) (presented as % of total VASP for pVASP, and fold change to vehicle-treated cells for pCREB and pHsp20). Human ASM cells were pre-incubated with 1 μM U0126 (MEK inhibitor) for 20 min, followed by 30 μM DGK inhibitor I for 15 min, and levels of PGE2 (E) and intracellular cAMP (F) were measured using ELISA-based assays. Human ASM cells stably expressing CRE-Luc were pre-incubated with 1 μM U0126 (MEK inhibitor) for 20 min, followed by 30 μM DGK inhibitor I for 6 h. Cells were lysed, and luminescence was recorded and plotted as fold change to luminescence detected in vehicle-treated cells (G). Data above is mean ± SEM of n=5 independent experiments; *p<0.05 using one-way-ANOVA with Bonferroni post-hoc analysis. Veh: Vehicle.

Role of PKA in DGK-mediated regulation of ASM contraction

Because our previous study demonstrated that DGK inhibition attenuates allergen-induced airway hyperresponsiveness (AHR) and inhibits agonist-induced airway contraction (Singh et al., 2019), we next assessed the effect of DGK inhibition and PKA activation on mechanisms of ASM contraction. PKA activation reduces the contraction of ASM by inhibiting MLC phosphorylation. Human ASM cells stably expressing GFP and PKI-GFP were incubated with DGK inhibitor I (30 μM, 15 min) followed by stimulation with histamine (1 μM, 10 min) (Fig. 6 A, B). Cell lysates obtained after histamine stimulation were used to assess phosphorylation of MLC by immunoblotting. Histamine stimulation resulted in increased phosphorylation of MLC, which was significantly attenuated by DGK inhibition in GFP expressing cells compared to PKI-GFP expressing cells (Fig. 6 B; p<0.05). These studies demonstrate that DGK-mediated inhibition of ASM contraction involves activation of the COX-PGE2-cAMP-PKA pathway via DAG-PKC/ERK (Fig. 7).

Figure 6: Role of PKA in DGK-mediated inhibition of MLC phosphorylation.

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Human ASM cells stably expressing PKI-GFP and GFP were pretreated with 30 μM DGK inhibitor I for 15 min, followed by stimulation with 1 μM histamine (His) for 10 min. Cells were lysed and immunoblotted for p-MLC, GFP, and β-actin (A). Graphical representation of mean ± SEM of the p-MLC (B). Data above is mean ± SEM of n=5 independent experiments; *p<0.05 using one-way-ANOVA with Bonferroni post-hoc analysis. Veh: Vehicle.

Figure 7: Model depicting the role of DGK in regulating ASM contraction via PKA activation.

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DGK inhibition increases DAG levels in human ASM cells, which activates PKC/MEK (ERK1/2)-mediated COX activation. This PKC/ERK-dependent posttranslational modulation of COX activity leads to PGE2 synthesis that activates Gs-coupled signaling (presumably through EP2/EP4), leading to an increase in cAMP level and PKA activation. PKA activation negatively regulates MLC phosphorylation required for cross bridging with actin and thus reducing contraction.

Discussion

Recently we showed that inhibition of DGK or genetic ablation reduced contraction of the airways and protected against allergen-induced airway hyperresponsiveness (Singh et al., 2019). However, the mechanism by which DGK inhibition attenuates contractile signaling in ASM is not well established. PKA plays a central role in inhibiting ASM contraction by relaxing agents such as agonists of beta 2 adrenoceptors (β2ARs) (Morgan et al., 2014). Although DGK participates in the pathway mediating pro-contractile signaling in ASM, we considered a possible role of PKA activation in the inhibitory effect of DGK inhibition on ASM contraction. Our findings demonstrate that DGK inhibition leads to enhanced PKC/ERK signaling, promoting cyclooxygenase (COX)-mediated production of PGE2, and the increased PGE2 promotes bronchoprotection via activation of PKA signaling in ASM (Figure 7).

Various regulatory mechanisms involving posttranslational modifications such as phosphorylation, glycosylation, nitrosylation, intracellular trafficking, ubiquitination, and ER-associated degradation (ERAD) have been proposed to underpin cellular COX activity upon acute activation (Alexanian & Sorokin, 2017; Kim, Huri, & Snyder, 2005). Increased DAG can activate both ERK and PKC. There is evidence for potential crosstalk between ERK and PKC to fine-tune their cellular responses to various perturbations to maintain homeostasis (Vezza, Habib, Li, Lawson, & FitzGerald, 1996). Further, while the mechanisms regulating COX degradation have been addressed extensively, much less is known about the precise posttranslational regulation that upregulates its enzymatic activity. For example, phosphorylation of COX-2 can increase PGE2 production (Alexanian, Miller, Chesnik, Mirza, & Sorokin, 2014). There are reports that certain protein-protein interactions can upregulate COX-2 activity (C. Yang & Sorokin, 2011) without change in COX-2 expression. For example, the 19 amino acid segment located at the COOH termini of COX-2 allows interaction with integrins in a Ras-ERK-dependent manner (Mbonye et al., 2006). These observations emanating from multiple studies consolidate our findings that acute DGK inhibition promotes COX activity in PKC/ERK-dependent manner, leading to inhibition of ASM contraction mediated by increased Gs-cAMP-PKA signaling.

cAMP signaling is a predominant regulator of physiological and pathophysiological processes in ASM. The beneficial effects mediated by elevated levels of cAMP in ASM are initiated following the binding of specific ligands to GPCRs of the Gs family, such as the β2AR on ASM cells (Kume, Hall, Washabau, Takagi, & Kotlikoff, 1994). Phosphodiesterase is also involved in regulating cAMP levels in ASM and thus maintaining airway tone (Rabe et al., 1993). Activation of cAMP signaling in ASM inhibits ASM contraction, proliferation, and migration (Goncharova et al., 2012; Morgan et al., 2014; Yan et al., 2011). Our previous studies have established that PKA plays a central role in the regulation of Gs-mediated inhibition of contraction and proliferation of ASM cells (Guo et al., 2005; Misior et al., 2008), although other effector molecules such as EPACs and AKAPs have been suggested (Horvat et al., 2012; Roscioni et al., 2011). The phosphorylation of numerous targets by PKA is known to either reduce intracellular calcium or calcium sensitivity in ASM (Endou et al., 2004; Y. Ito, Takagi, & Tomita, 1995; Mahn et al., 2010; Oguma et al., 2006), both of which lead to impairment of MLC phosphorylation resulting in reduced ASM contraction. Hsp20 is another important PKA target capable of inhibiting ASM contraction (Ba et al., 2009; Komalavilas et al., 2008). Our study using both molecular (heterologous expression of PKI) and pharmacological (H-89) approaches targeting PKA demonstrates that DGK inhibition reduces histamine-induced MLC phosphorylation via PKA activation. Although DGK is a key regulator of Gq signaling through its ability to promote the conversion of DAG into PA, our findings also reveal its ability to promote crosstalk between Gq and Gs signaling in ASM.

Another interesting finding from our study is related to the production of PGE2 due to the activation of the COX enzyme under the condition where DGK is inhibited. COX activity is the rate-limiting step for the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other metabolites that act as key regulators of airway cellular physiology (Laporte et al., 1999). COX-1 is expressed constitutively, whereas COX-2 is inducible and becomes elevated in several inflammatory conditions, including chronic airway diseases such as asthma (Khanapure, Garvey, Janero, & Letts, 2007; Pang, Holland, & Knox, 1998; Torres et al., 2008; Wenzel, 1997). Further, the prostanoid metabolism pathway is very complex and involves the release of a variety of mediators, which induce different signaling in various effector cells, thereby regulating pathophysiological outcomes in diseases. More specifically, we and others have shown that any change in the levels/activity of COX-2 and PGE2 can modulate ASM contractility (Gavett et al., 1999; Pang et al., 1998; Peebles et al., 2002; Sharma et al., 2012; Torres et al., 2008) presumably through EP2/4 receptors expressed on ASM cells (Backlund, Mann, & Dubois, 2005; Park et al., 2010; Ruan et al., 2008). Our current study demonstrates the novel finding that DGK may regulate COX-2 induction in ASM cells. It has been shown that DGKε plays an important role in the induction of COX-2 in other organs such as the kidney, brain, and adipose tissue (Lukiw et al., 2005; Nakano et al., 2020; Zhu et al., 2016) but it is not clear how DGK isoforms modulate COX-2 expression in ASM. In this study, we provide evidence that DGK inhibition enhances PKC and ERK activation, leading to the induction of COX-2 in ASM. MAP kinases in many cell types are also involved in COX-2 induction by cytokines, lipopolysaccharide, or growth factors. It has been shown that ERK activation is required for cytokine-induced COX-2 expression and PGE2 production in ASM cells (Belvisi, Saunders, Yacoub, & Mitchell, 1998; Laporte et al., 1999; Pascual et al., 2001). ERK MAP kinase activation induces AA metabolism and the formation of PGs such as PGE2 by phosphorylation of cPLA2 (Davis, 1993). As highlighted above, COX-2 induction is observed in many pathologies, our results in this study led us to believe that constitutive DGK activity keeps COX-2 restrained, thereby representing a break on its activity. Another possibility that needs to be tested is the role of subcellular compartments in regulating COX-2 activity under DGK inhibition. Our findings suggest that DGK inhibition releases homeostatic COX inhibition leading to increased PGE2 production in an ERK MAP kinase-dependent manner leading to bronchoprotection.

DGK inhibition affects cellular levels of DAG and PA, with a resultant increase in various species of DAG molecules. These subclasses of lipids have been identified as potential molecular activators of downstream signaling, most importantly, PKC activation. Correlation analysis has shown interconnections between DGK and PKC isoforms in distinct signaling nodes (Chianale et al., 2010; Kai et al., 2009; Rainero et al., 2014). For example, DGKα act as a central element of a lipid signaling pathway to regulate PKC that allows spatiotemporal signaling in epithelial cells (Chianale et al., 2010) and matrix invasion of carcinoma cells (Rainero et al., 2014). Therefore, cellular DAG and PA equilibrium could be a tool to fine-tune DGK and PKC downstream signaling (Mochly-Rosen, Das, & Grimes, 2012). Previous studies have shown that PKC signaling activated by DAG is also involved in regulating MAP kinases activation, including ERK1/2 (Zhang, Cardell, Edvinsson, & Xu, 2013). As described above, ERK MAP kinase has been shown to play a role in COX-2 induction in ASM cells. In agreement with these published works, our findings suggest that inhibition of DGK results in increased levels of DAG, resulting in increased activation of PKC-ERK that enhances COX-2 activity in ASM cells.

We show that DGK inhibition reduced MLC phosphorylation under contractile agonist stimulation. PKC is an important promotor of ASM contraction (Dempsey et al., 2000) wherein PKC can phosphorylate many essential contractile proteins in ASM, and its activation appears to be more involved in the sustained rather than the initial phases of ASM contraction (Sakai, Yamamoto, Chiba, & Misawa, 2009). Conversely, our findings suggest that DGK inhibition (which should promote PKC activity) attenuates agonist-induced contraction of ASM. In this context, activation of DGK and PKC are complex and bidirectional and are dictated by the levels of phospholipids. For example, DAG activates PKC, which in turn activates DGKζ isoform by phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) domain. Further, DGK interaction with PKC reduces its activity and promotes DAG metabolism (B. Luo, Prescott, & Topham, 2003) thereby optimizing PKC activity (Lee, Yamamoto, Kim, & Tanaka-Yamamoto, 2015; B. Luo et al., 2003; Olenchock et al., 2006). PKC is known to modulate the function of multiple additional proteins and our results in this study suggest that regulation of DGK-mediated contraction of ASM involves activation of COX-2 albeit involving activation of PKC. The kinetics (time course and magnitude) of activation of other signal transduction pathways and functions such as cell proliferation and migration in ASM cells under DGK inhibition need additional studies. In summary, our results provide additional control of ASM contraction through DGK by directly regulating COX-2 dependent activation of PGE2.

Acknowledgments

This study was funded by the National Institutes of Heart, Lung, and Blood Institute Grant HL146645 (to D. A. D. and T.K), HL137030 (to D.A.D), and HL058506 (to R.B.P.).

Footnotes

Conflict of interests

The authors declare no conflict of interest for the current study.

Data availability statement

All the data in the article are available upon reasonable request.

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