Tumor necrosis factor α (TNFα), a proinflammatory cytokine, plays a significant role in mediating the effects of acute inflammation in response to allergens, pollutants, and respiratory infections. ...Previously, we showed that acute exposure to TNFα induces mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with increased expression of dynamin-related protein 1 (DRP1). Phosphorylation of DRP1 at serine 616 (pDRP1
) promotes its translocation and binding to the outer mitochondrial membrane (OMM) and mediates mitochondrial fragmentation. Previously, we reported that TNFα exposure triggers protein unfolding and triggers an endoplasmic reticulum (ER) stress response involving phosphorylation of inositol-requiring enzyme 1α (pIRE1α) at serine 724 (pIRE1α
) and subsequent splicing of X-box binding protein 1 (XBP1s) in hASM cells. We hypothesize that TNFα-mediated activation of the pIRE1α
/XBP1s ER stress pathway in hASM cells transcriptionally activates genes that encode kinases responsible for pDRP1
phosphorylation. Using 3-D confocal imaging of MitoTracker green-labeled mitochondria, we found that TNFα treatment for 6 h induces mitochondrial fragmentation in hASM cells. We also confirmed that 6 h TNFα treatment activates the pIRE1α/XBP1s ER stress pathway. Using in silico analysis and ChIP assay, we showed that CDK1 and CDK5, kinases involved in the phosphorylation of pDRP1
, are transcriptionally targeted by XBP1s. TNFα treatment increased the binding affinity of XBP1s on the promoter regions of CDK1 and CDK5, and this was associated with an increase in pDRP1
and mitochondria fragmentation. This study reveals a new underlying molecular mechanism for TNFα-induced mitochondrial fragmentation in hASM cells.
Airway inflammation is increasing worldwide. Proinflammatory cytokines mediate an adaptive mechanism to overcome inflammation-induced cellular stress. Previously, we reported that TNFα mediates hASM cellular responses, leading to increased force and ATP consumption associated with increased O
consumption, and oxidative stress. This study indicates that TNFα induces ER stress, which induces mitochondrial fragmentation via pIRE1α
/XBP1s mediated CDK1/5 upregulation and pDRP1
phosphorylation. Mitochondrial fragmentation may promote hASM mitochondrial biogenesis to maintain healthy mitochondrial pool.
The effects of airway inflammation on airway smooth muscle (ASM) are mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα). In this review article, we will provide a ...unifying hypothesis for a homeostatic response to airway inflammation that mitigates oxidative stress and thereby provides resilience to ASM. Previous studies have shown that acute exposure to TNFα increases ASM force generation in response to muscarinic stimulation (hyper-reactivity) resulting in increased ATP consumption and increased tension cost. To meet this increased energetic demand, mitochondrial O
consumption and oxidative phosphorylation increases but at the cost of increased reactive oxygen species (ROS) production (oxidative stress). TNFα-induced oxidative stress results in the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and mitochondria of ASM. In the ER, TNFα selectively phosphorylates inositol-requiring enzyme 1 alpha (pIRE1α) triggering downstream splicing of the transcription factor X-box binding protein 1 (XBP1s); thus, activating the pIRE1α/XBP1s ER stress pathway. Protein unfolding in mitochondria also triggers an unfolded protein response (
UPR). In our conceptual framework, we hypothesize that activation of these pathways is homeostatically directed towards mitochondrial remodeling via an increase in peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression, which in turn triggers: (1) mitochondrial fragmentation (increased dynamin-related protein-1 (Drp1) and reduced mitofusin-2 (Mfn2) expression) and mitophagy (activation of the Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/Parkin mitophagy pathway) to improve mitochondrial quality; (2) reduced Mfn2 also results in a disruption of mitochondrial tethering to the ER and reduced mitochondrial Ca
influx; and (3) mitochondrial biogenesis and increased mitochondrial volume density. The homeostatic remodeling of mitochondria results in more efficient O
consumption and oxidative phosphorylation and reduced ROS formation by individual mitochondrion, while still meeting the increased ATP demand. Thus, the energetic load of hyper-reactivity is shared across the mitochondrial pool within ASM cells.
Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα ...activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (ER) stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells’ phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 μM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1αS724) were determined through Western blotting. The splicing of XBP1 mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1αS724 phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in XBP1s mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells.
Inflammatory airway diseases such as asthma affect more than 300 million people world-wide. Inflammation triggers pathophysiology via such as tumor necrosis factor α (TNFα) and interleukins (e.g., ...IL-13). Hypercontraction of airway smooth muscle (ASM) and ASM cell proliferation are major contributors to the exaggerated airway narrowing that occurs during agonist stimulation. An emergent theme in this context is the role of inflammation-induced endoplasmic reticulum (ER) stress and altered mitochondrial function including an increase in the formation of reactive oxygen species (ROS). This may establish a vicious cycle as excess ROS generation leads to further ER stress. Yet, it is unclear whether inflammation-induced ROS is the major mechanism leading to ER stress or the consequence of ER stress. In various diseases, inflammation leads to an increase in mitochondrial fission (fragmentation), associated with reduced levels of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2). Mitochondrial fragmentation may be a homeostatic response since it is generally coupled with mitochondrial biogenesis and mitochondrial volume density thereby reducing demand on individual mitochondrion. ER stress is triggered by the accumulation of unfolded proteins, which induces a homeostatic response to alter protein balance via effects on protein synthesis and degradation. In addition, the ER stress response promotes protein folding via increased expression of molecular chaperone proteins. Reduced Mfn2 and altered mitochondrial dynamics may not only be downstream to ER stress but also upstream such that a reduction in Mfn2 triggers further ER stress. In this review, we summarize the current understanding of the link between inflammation-induced ER stress and mitochondrial function and the role played in the pathophysiology of inflammatory airway diseases.
The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances ...mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.
During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca
concentration (Ca
), which leads to a contractile response ...excitation-contraction (E-C) coupling. Previously, the sensitivity of the contractile response of ASM to elevated Ca
(Ca
sensitivity) was assessed as the ratio of maximum force to maximum Ca
. However, this static assessment of Ca
sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured Ca
and isometric force responses to three concentrations of ACh (1, 2.6, and 10 μM). Both maximum Ca
and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca
sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced Ca
and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced Ca
and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of Ca
and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of Ca
, the extent of regulatory myosin light-chain (rMLC
) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of Ca
and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC
phosphorylation.
Enhanced airway contractility following inflammation by cytokines such as tumor necrosis factor alpha (TNFα) or interleukin-13 (IL-13) involves increased intracellular Ca(2+) (Ca(2+)(i)) levels in ...airway smooth muscle (ASM). In ASM, plasma membrane Ca(2+) fluxes form a key component of Ca(2+)(i) regulation. There is now growing evidence that the bidirectional plasma membrane Na(+)/Ca(2+) exchanger (NCX) contributes to ASM Ca(2+)(i) regulation. In the present study, we examined NCX expression and function in human ASM cells under normal conditions, and following exposure to TNFα or IL-13. Western blot analysis showed significant expression of the NCX1 isoform, with increased NCX1 levels by both cytokines, effects blunted by inhibitors of nuclear factor NF-κB or mitogen-activated protein kinase. Cytokine-mediated increase in NCX1 involved enhanced transcription followed by protein synthesis. NCX2 and NCX3 remained undetectable even in cytokine-stimulated ASM. In fura-2 loaded human ASM cells, NCX-mediated inward Ca(2+) exchange as well as outward exchange (measured as rates of change in Ca(2+)(i)) was elicited by altering extracellular Na(+) and Ca(2+) levels. Contribution of NCX was verified by measuring Na(+)(i) using the fluorescent Na(+) indicator SBFI. NCX-mediated inward exchange was verified by demonstrating prevention of rising Ca(2+)(i) or falling Na(+)(i) in the presence of the NCX inhibitor KBR7943. Inward exchange-mode NCX was increased by both TNFα and IL-13 to a greater extent than outward exchange. NCX siRNA transfection substantially blunted outward exchange and inward exchange modes. Finally, inhibition of NCX expression or function blunted peak Ca(2+)(i) and rate of fall of Ca(2+)(i) following histamine stimulation. These data suggest that NCX-mediated Ca(2+) fluxes normally exist in human ASM (potentially contributing to rapid Ca(2+) fluxes), and contribute to enhanced Ca(2+)(i) regulation in airway inflammation.
Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased ...PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREB
and pATF1
), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one-three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREB
, pATF1
, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREB
, pATF1
and PCG1α expression, with downstream transcriptional activation of
, and
We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREB
/pATF1
/PCG1α-mediated pathway.
Previously, we reported that in airway smooth muscle (ASM), the cytosolic Ca
2+
(Ca
2+
cyt
) and force response induced by acetyl choline (ACh) are increased by exposure to the pro-inflammatory ...cytokine tumor necrosis factor α (TNFα). The increase in ASM force induced by TNFα was not associated with an increase in regulatory myosin light chain (rMLC
20
) phosphorylation but was associated with an increase in contractile protein (actin and myosin) concentration and an enhancement of Ca
2+
dependent actin polymerization. The sensitivity of ASM force generation to elevated Ca
2+
cyt
(Ca
2+
sensitivity) is dynamic involving both the shorter-term canonical calmodulin-myosin light chain kinase (MLCK) signaling cascade that regulates rMLC
20
phosphorylation and cross-bridge recruitment as well as the longer-term regulation of actin polymerization that regulates contractile unit recruitment and actin tethering to the cortical cytoskeleton. In this study, we simultaneously measured Ca
2+
cyt
and force responses to ACh and explored the impact of 24-h TNFα on the dynamic relationship between Ca
2+
cyt
and force responses. The temporal delay between the onset of Ca
2+
cyt
and force responses was not affected by TNFα. Similarly, the rates of rise of Ca
2+
cyt
and force responses were not affected by TNFα. The absence of an impact of TNFα on the short delay relationships between Ca
2+
cyt
and force was consistent with the absence of an effect of Ca
2+
cyt
and force on rMLC
20
phosphorylation. However, the integral of the phase-loop plot of Ca
2+
cyt
and force increased with TNFα, consistent with an impact on actin polymerization and, contractile unit recruitment and actin tethering to the cortical cytoskeleton.
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