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Role of superoxide in apoptosis induced by growth factor withdrawal
W Lieberthal1, V Triaca, J S Koh
1Renal Section, Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118, USA.
Abstract:
We have examined the role of reactive oxygen species (ROS) in apoptosis induced by growth factor deprivation in primary cultures of mouse proximal tubular (MPT) cells. When confluent monolayers of MPT cells are deprived of all growth factors, the cells die by apoptosis over a 10- and 14-day period. Both epidermal growth factor (EGF) and high-dose insulin directly inhibit apoptosis of MPT cells deprived of growth factors. Growth factor deprivation results in an increase in the cellular levels of superoxide anion while apoptosis of MPT cells induced by growth factor withdrawal is inhibited by a number of antioxidants and scavengers of ROS. Growth factor deprivation also results in activation of caspase activity, which is inhibited by EGF and high-dose insulin as well as by the ROS scavengers and antioxidants that inhibit apoptosis. The cell-permeant caspase inhibitor, z-Val-Ala-Asp-CH2F (zVAD-fmk), prevents the increase in caspase activity and markedly inhibits apoptosis induced by growth factor deprivation. However, zVAD-fmk had no effect on the increased levels of superoxide associated with growth factor deprivation. Thus we provide novel evidence that ROS play an important role in mediating apoptosis associated with growth factor deprivation. ROS appear to act upstream of caspases in the apoptotic pathway. We hypothesize that oxidant stress, induced by growth factor withdrawal, represents a signaling mechanism for the default pathway of apoptosis.
Insights
Reactive oxygen species (ROS) mediate apoptosis in mouse proximal tubular cells deprived of growth factors. ROS act upstream of caspases, suggesting oxidant stress signals this cell death pathway.
Area of Science:
- Cell Biology
- Biochemistry
- Renal Physiology
Background:
- Growth factor deprivation induces apoptosis in primary mouse proximal tubular (MPT) cells.
- Epidermal growth factor (EGF) and insulin inhibit this apoptosis.
- The precise mechanisms, particularly the role of reactive oxygen species (ROS), remain unclear.
Purpose of the Study:
- To investigate the role of ROS in apoptosis of MPT cells induced by growth factor deprivation.
- To determine the relationship between ROS, caspases, and apoptosis in this model.
Main Methods:
- Primary cultures of MPT cells were deprived of growth factors.
- Cellular ROS levels (superoxide anion) and caspase activity were measured.
- The effects of antioxidants, ROS scavengers, EGF, insulin, and a caspase inhibitor (zVAD-fmk) on apoptosis were assessed.
Main Results:
- Growth factor deprivation increased cellular superoxide levels and caspase activity.
- Antioxidants, ROS scavengers, EGF, and insulin inhibited apoptosis and caspase activation.
- The caspase inhibitor zVAD-fmk inhibited apoptosis but did not affect elevated superoxide levels.
Conclusions:
- ROS play a critical role in mediating apoptosis induced by growth factor deprivation in MPT cells.
- ROS appear to function upstream of caspases in the apoptotic signaling pathway.
- Oxidant stress resulting from growth factor withdrawal may serve as a signaling mechanism for default apoptosis.