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CD44 induced enhancement of phosphatase activity and calcium influx: Modifications of EGR-1 expression and cell
Ronny R Racine1, Nathan A Manalo2, Jessica M F Hall1
1Department of Cell Biology and Immunology, University of North Texas Health Science Center, 3500, Camp Bowie Boulevard, Fort Worth TX 76104, United States.
Abstract:
The purpose of this study was to investigate how CD44 impaired Akt phosphorylation, EGR-1 expression and cell proliferation. E6.1 Jurkat cells, which lack endogenous CD44 expression, were engineered to express CD44. Previously we showed that Akt is hypophosphorylated, EGR-1 expression is reduced and proliferation is impaired in CD44 expressing E6.1 Jurkat cells. The cell cycle was studied using flow cytometry and the role of calcium (Ca2+) in Akt phosphorylation and EGR-1 expression was investigated using Western blotting. Phosphatase activity was assessed using a commercially available kit. CD44 expressing cells showed disruption at the G1 to S transition. Chelation of Ca2+ from the culture media impaired Akt phosphorylation and EGR-1 expression in both CD44 expressing cells and the open vector control. Moreover, Ni2+ disrupted cell proliferation in both cell types suggesting Ca2+ import through calcium release activated calcium channels (CRAC). Staining of cells with fura-2 AM showed significantly higher Ca2+ in CD44 expressing cells as compared with the vehicle control. Finally, non-calcium mediated phosphatase activity was significantly greater in CD44 expressing cells. We propose that the enhanced phosphatase activity in the CD44 cells increased the dephosphorylation rate of Akt; at the same time, the increased intracellular concentration of Ca2+ in the CD44 cells ensured that the phosphorylation of Akt remains intact albeit at lower concentrations as compared with the vector control. Reduced Akt phosphorylation resulted in lowered expression of EGR-1 and hence, reduced the cell proliferation rate.
Insights
CD44 expression in Jurkat cells impairs cell proliferation by disrupting the cell cycle and reducing Akt phosphorylation. This is linked to increased phosphatase activity and altered calcium signaling, impacting EGR-1 expression.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- CD44 is a cell surface glycoprotein involved in cell adhesion and migration.
- Dysregulation of CD44 has been implicated in various cancers.
- The precise mechanisms by which CD44 influences intracellular signaling pathways remain under investigation.
Purpose of the Study:
- To elucidate how CD44 expression affects Akt phosphorylation, EGR-1 expression, and cell proliferation.
- To investigate the role of calcium (Ca2+) in mediating these CD44-dependent effects.
- To explore the involvement of phosphatase activity in the observed signaling alterations.
Main Methods:
- Engineered E6.1 Jurkat cells to express CD44.
- Utilized flow cytometry for cell cycle analysis.
- Employed Western blotting to assess Akt phosphorylation and EGR-1 expression.
- Measured phosphatase activity using a commercial kit.
- Investigated calcium's role using Ca2+ chelation and Ni2+ treatment.
- Quantified intracellular Ca2+ levels with fura-2 AM staining.
Main Results:
- CD44-expressing cells exhibited cell cycle disruption at the G1 to S transition.
- Ca2+ chelation impaired Akt phosphorylation and EGR-1 expression in both CD44+ and control cells.
- Ni2+ treatment disrupted cell proliferation, suggesting calcium release-activated calcium channel (CRAC) involvement.
- CD44-expressing cells showed significantly higher intracellular Ca2+ levels.
- Non-calcium-mediated phosphatase activity was markedly greater in CD44-expressing cells.
Conclusions:
- Enhanced phosphatase activity in CD44-expressing cells increases Akt dephosphorylation.
- Elevated intracellular Ca2+ in CD44+ cells partially maintains Akt phosphorylation but at reduced levels.
- Reduced Akt phosphorylation leads to decreased EGR-1 expression and consequently, impaired cell proliferation.
- CD44 influences cell proliferation through modulation of calcium signaling and phosphatase activity, impacting key signaling proteins.
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