Abstract
Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.
Highlights
Recent studies have shown that annexin V, a cytosolic protein that binds to membranes in the presence of calcium, binds to the cytoplasmic domain of 5 integrin and to active protein kinase C ␣ (PKC␣)2 and that these interactions play a key role in the regulation of apoptosis of endothelial cells [11, 12]
5 integrin interactions are involved in mediating apoptotic events in growth plate chondrocytes, we used a peptide mimic of annexin V
We provide evidence that the balance between annexin V/5 integrin and annexin V/PKC␣ interactions plays a role in the regulation of growth plate chondrocyte apoptosis
Summary
Treatment of growth plate chondrocytes with chondrocytes in culture (Fig. 1B), confirming previous findings 12 M Pen-VVISYSMPD for 24 h led to a notable increase of showing an interaction between annexin V and the 5 integrin caspase-3 activity when compared with untreated cells or cells subunit [11]. Pen-VVISYSMPD treatment resulted in ϳ5-fold increase of Transfecting cells with siRNA specific for annexin V or treat- caspase-3 activity in growth plate chondrocytes when compared with the activity of untreated cells
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