ROLE OF INTRACELLULAR CALCIUM IN

GLUCOCORTICOID-EVOKED

LYMPHOID CELL APOPTOSIS

Devin Morris

California State University, Northridge

Apoptosis

Release ofcyctochrome c

EffectorCaspases

ApoptosisStimuli/Death signals

Apoptosomeformation

Activation of caspase cascade

Apaf-1 Caspase 9

Proliferation Cell Population Cell Death

• Development• Homeostasis• Disease

Physiological roles of apoptosis

R IPC

AB

C

IPCR

IPC

R

RR

RR

ligand receptor

interactingproteins

coactivator complex

basal trans. factors

1

2

3

4

Glucocorticoid Pathway

Susceptibility of CEM Cell Clones to GC-Evoked Apoptosis

GC-induced Increase in Intracellular Calcium in GC-Susceptible CEM cells

Time- & GC-Dose-Dependent Increase in [Ca+2]i in CEM-C7-14 Cells

EtOH 100 nM Dex 1M Dex

24 h

ou

rs48

ho

urs

4.4% 6% 14.1%

5% 24.8% 62.1%

A B C

D E F

Modulators of [Ca+2]i Levels Influence GC-Evoked Death of CEM Cells

EGTA Suppresses GC-Evoked Increase in [Ca+2]i Levels in CEM-C7-14 Cells

EtOH 100 nM Dex 1M Dex

Eth

ano

l20

M

EG

TA

A B C

D E F

4.4% 16.4% 35%

2% 12.4% 23.1%

Glucocorticoid Signaling Pathway

GC [Ca+2]i

(Release fromIntracellular

Stores)

TMB-8

Ca+2-CaM

Binding and Activation

W7 orcalmidazolium

chlorideActivation of

CAMK II

KN-62 orKN-93

Activation of Calcineurin

FK-506 or Cyclosporine A

GC [Ca+2]i

(Release fromIntracellular

Stores)

TMB-8

Ca+2-CaM

Binding and Activation

W7 orcalmidazolium

chlorideActivation of

CAMK II

KN-62 orKN-93

Activation of Calcineurin

FK-506 or Cyclosporine A

Pathway Inhibitors

Normal Pathway

Inhibition of Calmodulin Protects CEM-C7-14 Cells from GC-evoked Death

Inhibition of Calmodulin Kinase II Protects CEM-C7-14 Cells from GC-evoked Death

Inhibition of Calcineurin Protects CEM-C7-14 Cells from GC-evoked Death

Conclusions

• GCs increase [Ca+2]i levels only in the GC-susceptible CEM-C7-14 cell line in a dose dependent manner; not in the GC-resistant sister cell line, CEM-C1-15.

• Calcium chelation by either BAPTA or EGTA protected CEM-C7-14 cells from GC-evoked apoptosis, in conjunction with a reduction in the amount of free [Ca+2]i.

• The calcium ionophore A23187 causes sensitization of CEM-C1-15 cells to GC-evoked apoptosis.

• Inhibition of calmodulin, calmodulin kinase II or calcineurin, all intermediates in the calcium signaling pathway, impart varying degrees of protection to CEM-C7-14 cells from GC-evoked apoptosis.

• Our data demonstrate a clear correlation between calcium signaling and GC-evoked apoptosis

Future Goals

• Further studies will aim to understand Ca+2-dependent changes in gene regulation that contribute to apoptosis. Candidate genes such as the transcriptional repressor E4BP4, and its downstream targets are being studied.

• Our ultimate goal is to understand the molecular pathway for apoptosis in T-lymphoid cells as well as in other physiologically relevant models for apoptosis, such as osteoblasts, keratinocytes and macrophages.

Acknowledgements

Funded by grants from the NIH MBRS-SCORE Program, the CSUN Office of Graduate Studies, Research and International Programs, and the CSUN College of Science & Mathematics.

• Dr. Rheem Medh

• Saul Priceman

• Dr. Carol Shubin

• NASA CSUN/JPL Pair Program