CD44 induced enhancement of phosphatase activity and calcium influx: Modifications of EGR-1 expression and cell proliferation

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.

Keywords: Acute Lymphoblastic Leukemia; CD44; Calcium; Jurkat; Proliferation.

Fig. 1

CD44 expressing Jurkat T cells are arrested in phase G₁. (A) Cell cycle analysis was performed via propidium iodide staining and flow cytometry. Results show representative data from four repeated experiments. (B) BrdUrd incorporation was measured in cells synchronized in the G₁ phase of the cell cycle after being allowed to proliferate for 30 min. Results show representative data from four repeated experiments. Asterisks indicated statistical significance as assessed using the 2-tailed Student's t-test (*p<0.05, **p<0.01).

Fig. 2

CD44 expression induces calcium influx. Resting intracellular calcium concentration was assessed by ratiometric imaging of fura-2-AM loaded cells. Multiple images were taken for ratiometric assessment. The asterisk indicates the statistical significance using the 2-tailed Student's t-test (n=40 cells; **p<0.01).

Fig. 3

CD44 expression induced Ca²⁺ influx uses CRAC channels to regulate proliferation and Ca²⁺ is required for proper cell cycling. (A) Cell proliferation with extracellular calcium, without extracellular calcium, and with Ni²⁺, an inhibitor of CRAC channels under normal extracellular calcium levels. Results are normalized to percent of the vector control cells with calcium. (B) Cell cycle analysis was performed on CD44 expressing cells via propidium iodide staining and flow cytometry after incubation in calcium containing (open bars) or calcium free (closed bars) media for 24 h after synchronization. Asterisks indicate the statistical significance using the 2-tailed Student's t-test (*p<0.05).

Fig. 4

EGR-1 expression is regulated by Ca²⁺. (A) Western blot for EGR-1 expression with and without calcium. A representative blot is shown as the left panel and cumulative densitometry values are shown as the right panel. (B) Cell proliferation before, during, and after a 24 h period without extracellular calcium. Results are normalized to percent of control cell line with calcium. There was no difference between Vector and CD44 cell lines without calcium (p>0.05). (C) Western blotting for EGR-1 expression before removal of extracellular calcium, after one day of rest following 24 h without extracellular calcium, and after four days of rest following 24 h without extracellular calcium. A representative blot is shown as the left panel and cumulative densitometry values are shown as the right panel. Densitometry values from three different Western blots were used to calculate the means ± SD. Asterisks indicated statistical significance using the 2-tailed Student's t-test (A and B) or the ANOVA with Turkey's multiple comparisons test (C) (*p<0.05;**p<0.01).

Fig. 5

CD44 regulates EGR-1 through Ca²⁺ signaling and the Akt pathway. (A) Western blot for total Akt and phosphorylated Akt with and without extracellular calcium. A representative blot is shown as the left panel and cumulative densitometry values are shown as the right panel. (B) Western blot for EGR-1 expression with extracellular calcium (EC calcium), without EC calcium, with an inhibitor of Akt activation, and without EC calcium and an Akt inhibitor. A representative blot is shown as the left panel and cumulative densitometry values are shown as the right panels. Densitometry values from three different Western blots were used to calculate the means±SD. Asterisks indicated statistical significance using the 2-tailed Student's t-test (A) or the ANOVA with Turkey's multiple comparisons test (B) (*p<0.05).

Fig. 6

CD44 increases phosphatase activity in E6.1 Jurkat cells. (A) A phosphatase activity assay specific for PP1, PP2A, calcineurin, and PP2C. Total represents all measured phosphatase activity. Calcium Independent (Calcium Ind.) is the activity of PP1, PP2A, and PP2C. CaN is the activity of just calcineurin. (B) Western blot for total Akt and phosphorylated Akt with cantharidic acid. The Western blot was repeated 3 times with similar results. (C) Proliferation of cells with different concentrations of cantharidic acid over 48 h. Results are normalized to untreated vector control cells. (D) JC-1 staining for mitochondrial depolarization as measured by flow cytometry (left panel) and spectrofluorometry (right panel). Results are expressed as the ratio of red mitochondrial associated dye to green cytosol associated dye. Asterisks indicated statistical significance using the 2-tailed Student's t-test (A) or the ANOVA with Turkey's multiple comparisons test (C) (*p<0.05).