Papers by William Gerthoffer
Cell Health and Cytoskeleton, 2009
HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to i... more HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM) we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ) inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1), and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200-500 kDa) to smaller complexes (60 kDa). The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway smooth muscle. HSP20 is upregulated by beta agonists and downregulated by proinflammatory cytokines. HSP20 is phosphorylated in vivo in a cAMP-dependent manner and the phosphorylated form promotes airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin.
Advances in experimental medicine and biology, 2021
Asthma is a chronic inflammatory obstructive lung disease that is stratified into endotypes. Th2 ... more Asthma is a chronic inflammatory obstructive lung disease that is stratified into endotypes. Th2 high asthma is due to an imbalance of Th1/Th2 signaling leading to abnormally high levels of Th2 cytokines, IL-4, IL-5, and IL-13 and in some cases a reduction in type I interferons. Some asthmatics express Th2 low, Th1/Th17 high phenotypes with or without eosinophilia. Most asthmatics with Th2 high phenotype respond to beta-adrenergic agonists, muscarinic antagonists, and inhaled corticosteroids. However, 5-10% of asthmatics are not well controlled by these therapies despite significant advances in lung immunology and the pathogenesis of severe asthma. This problem is being addressed by developing novel classes of anti-inflammatory agents. Numerous studies have established efficacy of targeting pro-inflammatory microRNAs in mouse models of mild/moderate and severe asthma. Current approaches employ microRNA mimics and antagonists designed for use in vivo. Chemically modified oligonucleot...
Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failur... more Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverses established arterial wall thickening, stiffening and hypercontractility. Therefore, new approaches are needed to achieve long-acting prevention and reversal of occlusive pulmonary vascular remodeling. Several promising new drug classes are emerging from better understanding of pulmonary vascular gene expression programs. In this review potential epigenetic targets for small molecules and oligonucleotides will be described. Most are in preclinical studies aimed at modifying growth of vascular wall cells in vitro or normalizing vascular remodeling in PAH animal models. Initial success with lung-directed delivery of oligonucleotides targeting microRNAs suggests other epigenetic mechanisms might also be suitable drug targets. Those targets include DNA methylation, prote...
Brian S. Comer, Blanca Camoretti-Mercado, Paul C. Kogut, Andrew J. Halayko, Julian Solway, and Wi... more Brian S. Comer, Blanca Camoretti-Mercado, Paul C. Kogut, Andrew J. Halayko, Julian Solway, and William T. Gerthoffer Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama; Center for Personalized Medicine and Genomics, Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida; Department of Medicine and Institute for Translational Medicine, University of Chicago, Chicago, Illinois; Departments of Physiology and Pathophysiology, and Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; and Department of Pediatrics, Institute of Translational Medicine, University of Chicago, Chicago, Illinois
American Journal of Physiology …, 1983
We have tested the hypothesis that phosphorylation of the 20,000-dalton myosin light chains (LC 2... more We have tested the hypothesis that phosphorylation of the 20,000-dalton myosin light chains (LC 20) in rabbit tracheal smooth muscle modulates cross-bridge kinetics and isotonic shortening velocity. The thin muscle [190 +/- 10 (SE) microns] allowed detection of rapid changes in carbachol-induced active stress development, LC 20 phosphorylation, and isotonic shortening velocities. Phosphorylation of the LC 20 in resting muscle was 0.12 +/- 0.04 mol Pi/mol LC 20. Carbachol (10(-5) M) increased the level of phosphorylation to 0.46 +/- 0.03 mol Pi/mol LC 20 within 30 s. Phosphorylation then declined significantly as steady-state active stress was reached. A positive correlation was always found between LC 20 phosphorylation and shortening velocity. This result supports the hypothesis that the level of myosin phosphorylation was related to the mean cross-bridge cycling rate rather than the number of cross bridges contributing to the developed stress. Dephosphorylation of LC 20 occurred at about the same rate as the decline in shortening velocity and stress upon stimulus washout.
International Journal of Molecular Sciences
Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failur... more Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverse established arterial wall thickening, stiffening, and hypercontractility. Therefore, new approaches are needed to achieve long-acting prevention and reversal of occlusive pulmonary vascular remodeling. Several promising new drug classes are emerging from a better understanding of pulmonary vascular gene expression programs. In this review, potential epigenetic targets for small molecules and oligonucleotides will be described. Most are in preclinical studies aimed at modifying the growth of vascular wall cells in vitro or normalizing vascular remodeling in PAH animal models. Initial success with lung-directed delivery of oligonucleotides targeting microRNAs suggests other epigenetic mechanisms might also be suitable drug targets. Those targets include DNA methylation...
Molecular Therapy - Nucleic Acids
American Journal of Physiology-Gastrointestinal and Liver Physiology
We have determined that serum source plays a critical role in optimizing conditions for an organ ... more We have determined that serum source plays a critical role in optimizing conditions for an organ culture model of canine proximal colon. Previous studies using equine serum in the medium have shown that some properties of canine colonic smooth muscle can be maintained in organ culture. However, many characteristics of the tissue were altered by the culture conditions. The aims of the present study were to determine whether serum isolated from canine blood would improve the preservation of physiological properties of canine proximal colon in organ culture. Strips of canine colonic smooth muscle were cultured in 10% canine serum medium, and electrical, mechanical, morphological, and molecular analyses were performed after 0, 3, and 6 days in culture. Unlike organ culture in equine serum, in which Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) expression declined, culture in canine serum maintained Na+-K+-ATPase expression, and resting membrane potential of smooth muscle cells along th...
Pulmonary pharmacology & therapeutics, Jan 8, 2018
Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role i... more Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role in airway diseases. This is partially regulated by p38 mitogen-activated protein kinase (MAPK), which further activates two closely related isoforms of the MAPK-activated protein kinases (MKs), MK2 and MK3. The MKs have similar substrate specificities but less is known about differences in their functional responses. This study was undertaken to identify differential downstream inflammatory targets of MK2 and MK3 signaling and assess cross-talk between the MAPK pathway and NF-κB signaling relevant to ASM function. Wild-type and kinase-deficient MK2 (MK2, MK2) and MK3 (MK3, MK3) were expressed in human ASM cells stimulated for 20 h with 10 ng/ml each interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Inflammatory mediator secretion was assessed by Luminex assays and ELISA. Signaling pathway activation was monitored by Western blotting. Expression of these MKs and st...
American journal of respiratory and critical care medicine, Jan 12, 2017
microRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of pa... more microRNAs (miRNAs) destabilize mRNA transcripts and inhibit protein translation. miR-145 is of particular interest in cystic fibrosis (CF) as it has a direct binding site in the 3'-untranslated region of CFTR and is upregulated by the CF genetic modifier TGF-β. miR-145 mediates TGF-β inhibition of CFTR synthesis and function in airway epithelia. Primary human CF (F508del homozygous) and non-CF airway epithelial cells (AECs) were grown to terminal differentiation at air-liquid interface on permeable supports. TGF-β (5 ng/ml), a miR-145 mimic (20 nM), and a miR-145 antagonist (20 nM) were used to manipulate CFTR function. In CF cells, lumacaftor (3 µM) and ivacaftor (10 µM) corrected mutant F508del CFTR. Quantification of CFTR mRNA, protein, and function utilized standard techniques. miR-145 is increased 4-fold in CF bronchoalveolar lavage fluid compared to non-CF (p<0.01) and increased 10-fold in CF primary AECs (p<0.01). Exogenous TGF-β doubles miR-145 expression (p<0.0...
Annals of the American Thoracic Society, 2016
American Journal of Physiology Lung Cellular and Molecular Physiology, May 1, 2007
Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as ... more Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M2 and M3 receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.
Airway smooth muscle (ASM) plays a pivotal role in modulating bronchomotor tone but also orchestr... more Airway smooth muscle (ASM) plays a pivotal role in modulating bronchomotor tone but also orchestrates and perpetuates airway inflammation and remodeling. Despite substantial research, there remain important unanswered questions. In 2006, the National Heart, Lung, and Blood Institute sponsored a workshop to define new directions in ASM biology. Important questions concerning the key functions of ASM include the following: Does developmental dysregulation of ASM function promote airway disease, what key signaling pathways in ASM evoke airway hyperresponsiveness in vivo, do alterations in ASM mass affect excitation-contraction coupling, and can ASM modulate airway inflammation and remodeling in a physiologically relevant manner? This workshop identified critical issues in ASM biology to delineate areas for scientific investigation in the identification of new therapeutic and diagnostic approaches in asthma, chronic obstructive pulmonary disease, and cystic fibrosis.
Molecular and Cellular Pharmacology, Feb 28, 2012
Advances in studies of microRNA (miRNA) expression and function in smooth muscles illustrate impo... more Advances in studies of microRNA (miRNA) expression and function in smooth muscles illustrate important effects of small noncoding RNAs on cell proliferation, hypertrophy and differentiation. An emerging theme in miRNA research in a variety of cell types including smooth muscles is that miRNAs regulate protein expression networks to fine tune phenotype. Some widely expressed miRNAs have been described in smooth muscles that regulate important processes in many cell types, such as miR-21 control of proliferation and cell survival. Other miRNAs that are prominent regulators of smooth muscle-restricted gene expression also have targets that control pluripotent cell differentiation. The miR-143~145 cluster which targets myocardin and Kruppel-like factor 4 (KLF4) is arguably the best-described miRNA family in smooth muscles with profound effects on gene expression networks that promote serum response factor (SRF)-dependent contractile and cytoskeletal protein expression and the mature contractile phenotype. Kruppel-family members KLF4 and KLF5 have multiple effects on cell differentiation and are targets for multiple miRNAs in smooth muscles (miR-145, miR-146a, miR-25). The feedback and feedforward loops being defined appear to contribute significantly to vascular and airway remodeling in cardiovascular and respiratory diseases. RNA interference approaches applied to animal models of vascular and respiratory diseases prove that miRNAs and RNA-induced silencing are valid targets for novel anti-remodeling therapies that alter pathological smooth muscle hyperplasia and hypertrophy.
The Faseb Journal, Apr 1, 2014
Circulation, Oct 31, 2006
The Faseb Journal, Apr 1, 2014
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Papers by William Gerthoffer