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TGF-β1 Mediates HASM Cell Hyporesponsiveness to β2-Agonist-Induced Relaxation
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A1223 - TGF-β1 Mediates HASM Cell Hyporesponsiveness to β2-Agonist-Induced Relaxation
Author Block: C. A. Ojiaku1, J. K. Williams2, R. A. Panettieri3; 1Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, United States, 2Drexel University, Philadelphia, PA, United States, 3Institute for Translational Medicine and Science, Rutgers University, New Brunswick, NJ, United States.
RATIONALE: Several cytokines modulate both HASM cell agonist-induced hyperresponsiveness and β2-agonist hyporesponsiveness in asthma. Whether transforming growth factor beta 1 (TGF-β1), which induces HASM cell shortening and hyperresponsiveness, modulates HASM cell relaxation remains unclear. We hypothesize that TGF-β1-induced HASM cell shortening is resistant to β2-agonist-mediated relaxation.
METHODS: Human precision-cut lung slices (hPCLS) were treated with TGF-β1 (100 ng/mL) overnight and constricted to >50% with the contractile agonist carbachol. Subsequently, bronchodilation to formoterol was detected in slices by analyzing changes in airway lumen area using a live-feed microscope and an Image Pro-Plus software macro. Serum-starved HASM cells were treated with TGF-β1 (10 ng/mL) overnight prior to stimulation with isoproterenol (ISO) (20 µM, 5 min) in the presence or absence of carbachol (Cch) (20 µM, 10 min). HASM cell protein was isolated and expression of phosphorylated myosin light chain (MLC), AKT, and ERK were determined by immunoblot. Intracellular cAMP levels were determined by chemiluminescent immunoassay in TGF-β1-treated cells following ISO (1 µM, 10 min) and/or Cch (20 µM, 12 min) stimulation.
RESULTS: Overnight TGF-β1 treatment induced a slight reduction in hPCLS airway lumen bronchodilation over that of control. ISO stimulation of HASM cells significantly decreased HASM cell MLC phosphorylation induced by Cch (48.85% ± 22.26; P=0.02), but not that induced by TGF-β1 (26.79% ± 17.1; P=0.44). Additionally, Cch stimulation following overnight TGF-β1 treatment induced a hyperresponsive phenotype in HASM cells that conferred resistance to ISO-induced MLC dephosphorylation. In TGF-β1 treated cells, ISO-induced intracellular cAMP levels were significantly decreased versus that of control. Furthermore, inhibition of phosphodiesterase activity with IBMX (500 µM) reduced the inhibitory effect of TGF-β1 on ISO-induced cAMP generation.
CONCLUSION: Our data suggest that TGF-β1 induces HASM cell β2-agonist hyporesponsiveness by modulating intracellular cAMP levels. The reversal of TGF-β1’s inhibitory effect on ISO-induced cAMP with IBMX suggests a role for phosphodiesterases in TGF-β1-induced modulation of HASM cell relaxation.
Author Block: C. A. Ojiaku1, J. K. Williams2, R. A. Panettieri3; 1Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, United States, 2Drexel University, Philadelphia, PA, United States, 3Institute for Translational Medicine and Science, Rutgers University, New Brunswick, NJ, United States.
RATIONALE: Several cytokines modulate both HASM cell agonist-induced hyperresponsiveness and β2-agonist hyporesponsiveness in asthma. Whether transforming growth factor beta 1 (TGF-β1), which induces HASM cell shortening and hyperresponsiveness, modulates HASM cell relaxation remains unclear. We hypothesize that TGF-β1-induced HASM cell shortening is resistant to β2-agonist-mediated relaxation.
METHODS: Human precision-cut lung slices (hPCLS) were treated with TGF-β1 (100 ng/mL) overnight and constricted to >50% with the contractile agonist carbachol. Subsequently, bronchodilation to formoterol was detected in slices by analyzing changes in airway lumen area using a live-feed microscope and an Image Pro-Plus software macro. Serum-starved HASM cells were treated with TGF-β1 (10 ng/mL) overnight prior to stimulation with isoproterenol (ISO) (20 µM, 5 min) in the presence or absence of carbachol (Cch) (20 µM, 10 min). HASM cell protein was isolated and expression of phosphorylated myosin light chain (MLC), AKT, and ERK were determined by immunoblot. Intracellular cAMP levels were determined by chemiluminescent immunoassay in TGF-β1-treated cells following ISO (1 µM, 10 min) and/or Cch (20 µM, 12 min) stimulation.
RESULTS: Overnight TGF-β1 treatment induced a slight reduction in hPCLS airway lumen bronchodilation over that of control. ISO stimulation of HASM cells significantly decreased HASM cell MLC phosphorylation induced by Cch (48.85% ± 22.26; P=0.02), but not that induced by TGF-β1 (26.79% ± 17.1; P=0.44). Additionally, Cch stimulation following overnight TGF-β1 treatment induced a hyperresponsive phenotype in HASM cells that conferred resistance to ISO-induced MLC dephosphorylation. In TGF-β1 treated cells, ISO-induced intracellular cAMP levels were significantly decreased versus that of control. Furthermore, inhibition of phosphodiesterase activity with IBMX (500 µM) reduced the inhibitory effect of TGF-β1 on ISO-induced cAMP generation.
CONCLUSION: Our data suggest that TGF-β1 induces HASM cell β2-agonist hyporesponsiveness by modulating intracellular cAMP levels. The reversal of TGF-β1’s inhibitory effect on ISO-induced cAMP with IBMX suggests a role for phosphodiesterases in TGF-β1-induced modulation of HASM cell relaxation.
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