ABSTRACT
Discoidin domain receptor (DDR)1 is an extracellular matrix (ECM)-sensing receptor tyrosine kinase, which is activated by collagen and expressed in bronchial epithelium. DDR1 is responsible for maintaining the normal structure of skin and kidney epithelia and we hypothesised that DDR1 plays a regulatory role in bronchial epithelial integrity by transducing signals from the airway ECM. Effects of DDR1 depletion were studied using RNA interference in primary human bronchial epithelial cells (HBECs) and BEAS-2B cells. The effects of overexpression of DDR1a and DDR1b in BEAS-2B cells were studied using a plasmid vector. We measured the effects on epithelial repair using a scratch wounding model, and levels of matrix metalloproteinases (MMPs) by gelatin zymography (MMP-2 and -9) and ELISA (MMP-7). We showed that knockdown of DDR1 slowed epithelial repair by 50%, which was associated with a reduction in levels of MMP-7, whilst DDR1 overexpression enhanced epithelial repair. DDR1 knockdown reduced proliferation of HBECs, but had no significant effect on adhesion to collagen I or other matrix substrates. These data suggest that ECM signalling via DDR1 regulates aspects of bronchial epithelial repair, integrity and MMP expression in the airways.
Subject(s)
Bronchi/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 7/metabolism , Matrix Metalloproteinase 9/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Adult , Aged , Asthma/enzymology , Asthma/pathology , Bronchi/pathology , Cell Adhesion , Cell Line , Cell Proliferation , Cells, Cultured , Collagen Type I/metabolism , Discoidin Domain Receptor 1 , Female , Gene Knockdown Techniques , Humans , Male , Middle Aged , Receptor Protein-Tyrosine Kinases/genetics , Smoking/metabolism , Wound Healing , Young AdultABSTRACT
Apoptosis is a physiological program for the deletion of cells in which caspases govern events leading to safe clearance by phagocytes. However, a growing weight of evidence now suggests that not all forms of programmed cell death are caspase-dependent. We now report a complete and constitutive but caspase-independent program for the specific phagocytic clearance of intact effete platelets, anucleated blood cells of critical importance in health and disease. Platelets aged in vitro not only exhibited increased expression of proapoptotic Bak and Bax but also evidenced constitutive diminution of function such as decreased aggregation to ADP, which was accelerated by culture in the absence of plasma. This abrogation of cell function in plasma-deprived platelets was associated with morphological and biochemical features similar to those of granulocyte apoptosis, that is, cytoplasmic condensation, plasma membrane changes including exposure of phosphatidylserine and the granule protein P-selectin, and recognition by phagocyte scavenger receptors. However, and in contrast with constitutive death of other inflammatory blood cells by apoptosis, these events were not affected by caspase inhibitors, nor was there evidence of caspase-3 activation either by hydrolysis of analog peptide substrates or Western blot analysis, serving to emphasize that neither programmed cell death nor clearance by phagocytes need involve caspases.
