Activation of p55 Tumor Necrosis Factor-α Receptor-1 Coupled to Tumor Necrosis Factor Receptor-Associated Factor 2 Stimulates Intercellular Adhesion Molecule-1 Expression by Modulating a Thapsigargin-Sensitive Pathway in Human Tracheal Smooth Muscle Cells
- Yassine Amrani,
- Aili L. Lazaar,
- Rebecca Hoffman,
- Kunjalata Amin,
- Samir Ousmer and
- Reynold A. Panettieri, Jr.
- Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
Abstract
Tumor necrosis factor-α (TNFα) stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) by activating the transcription factor nuclear factor-κB (NF-κB) in human airway smooth muscle (ASM) cells. This study characterizes the receptor involved as well as critical downstream signaling events mediating cytokine-induced NF-κB activation and ICAM-1 expression. TNFα stimulation for 1 to 4 h induced ICAM-1 expression in human ASM cells. This rapid TNFα-induced ICAM-1 expression enhanced T-lymphocyte adhesion to ASM cells, which was inhibited by anti-ICAM-1 antibodies. Using immunostaining, we demonstrated that TNFα receptors TNFR1 and TNFR2 are expressed on native human tracheal smooth muscle. Treatment of cells with htr-9, an antibody that specifically activates TNFR1, also stimulated expression of ICAM-1 mRNA and protein. Utr-1, a blocking antibody to TNFR2, did not affect TNFα-mediated ICAM-1 expression. Both TNFα and htr-9 increased luciferase activity in ASM cells transfected with a NF-κB reporter plasmid. Overexpression of a dominant negative TNF receptor-associated factor 2 construct, lacking the NH2-terminal RING finger, completely abrogated both TNFα- and htr-9-mediated increases in NF-κB reporter activity. Thapsigargin, an agent that depletes intracellular calcium stores, abrogated both cytokine-mediated NF-κB-dependent ICAM-1 mRNA transcription and protein expression but had no effect on IκB degradation. In addition, chelating cytosolic calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester also inhibited cytokine TNFα-induced ICAM-1 expression. These data suggest that TNFR1, through a TNF receptor-associated factor 2-NF-κB signaling pathway, mediates TNFα-induced expression of ICAM-1 on ASM cells by involving a thapsigargin-sensitive signaling pathway.
Footnotes
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Dr. Yassine Amrani, University of Pennsylvania Medical Center, Pulmonary, Allergy and Critical Care Division, 848 Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160. E-mail: amrani{at}mail.med.upenn.edu
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This study was supported by Grants HL03202 (to A.L.L.), McCabe Research Fund of the University of Pennsylvania (to A.L.L.), HL55301 (to R.A.P.), AI40203 (to R.A.P.), American Lung Association Career Investigator Award (to R.A.P.), and HL64063 (to R.A.P.).
- Abbreviations:
- ASM
- airway smooth muscle
- TNFα
- tumor necrosis factor-α
- IL1β
- interleukin-1β
- ICAM-1
- intercellular adhesion molecule-1
- VCAM-1
- vascular cell adhesion molecule-1
- NF-κB
- nuclear factor-κB
- TRADD
- TNF receptor-associated death domain
- TNFR
- TNF receptor
- TRAF2
- TNF receptor-associated factor 2
- DN
- dominant negative
- BAPTA-AM
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester
- FBS
- fetal bovine serum
- RT-PCR
- reverse transcription-polymerase chain reaction
- CHX
- cycloheximide
- IκB
- inhibitor of κB
- RANTES
- regulated upon activation normal T cell expressed and secreted
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- Received December 29, 1999.
- Accepted March 28, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
