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Cigarette smoke extract increases C5a receptor expression in human bronchial epithelial cells
Diane S Allen-Gipson1, Anthony A Floreani, Art J Heires
1Pulmonary, Critical Care, and Sleep Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, 985815 Nebraska Medical Center, Omaha, NE 68198-5815, USA. dallengipson@unmc.edu
The Journal of Pharmacology and Experimental Therapeutics
|April 22, 2005
Summary
Cigarette smoke extract (CSE) increases the number of C5a anaphylatoxin receptors (C5aR) on bronchial cells. Protein kinase C-alpha (PKC-alpha) activity appears to modulate C5aR binding, suggesting a role in airway inflammation.
Area of Science:
- Immunology
- Cell Biology
- Respiratory Medicine
Background:
- Cigarette smoke extract (CSE) is known to induce inflammatory responses in the airways.
- The C5a anaphylatoxin receptor (C5aR) plays a role in inflammatory processes.
- Human bronchial epithelial cells (HBECs) are a primary target of inhaled irritants like CSE.
Purpose of the Study:
- To quantify C5aR expression and binding affinity on HBECs exposed to CSE.
- To investigate the role of protein kinase C-alpha (PKC-alpha) in modulating C5aR availability and binding.
- To explore the functional relationship between CSE, C5aR, and PKC-alpha in bronchial epithelial cells.
Main Methods:
- Whole-cell saturation binding studies using radiolabeled C5a to determine C5aR number and affinity.
- Competitive binding assays to identify different classes of C5a binding sites.
- Transfection of BEAS-2B cells with wild-type (WT) or dominant-negative (DN) PKC-alpha.
- Western blot analysis to confirm protein expression and functional assays to assess PKC activity.
- Inhibition of PKC-alpha using Gö6976 to evaluate its effect on C5aR binding.
Main Results:
- CSE exposure significantly increased C5aR expression and binding sites on HBECs, with detectable binding only after CSE treatment.
- Competitive binding studies revealed two classes of C5a binding sites with distinct affinities.
- PKC-alpha activity was found to be crucial for C5aR binding; inhibition or dominant-negative expression altered C5a binding affinity.
- Pretreatment with a PKC-alpha inhibitor (Gö6976) shifted the C5a binding curve in WT cells, indicating modulation of C5aR binding.
Conclusions:
- C5aR is expressed on HBECs and its availability is increased by CSE exposure.
- PKC-alpha plays a significant role in regulating C5aR binding affinity and availability in bronchial epithelial cells.
- These findings suggest a novel mechanism involving PKC-alpha in CSE-induced airway inflammation via C5aR modulation.