흰쥐 C6 신경교종 세포에서 Fluoxetine의 전처리 시간에 따른 Dexamethasone-매개 열충격 단백질 70의 발현 억제 = Fluoxetine pretreatment exert time-dependent inhibits the dexamethasone-mediated heat shock protein 70 expression in rat C6 glioma cells

다국어 초록 (Multilingual Abstract)

Objective : The aim of this study was to examine the effect of dexamethasone and fluoxetine on the expression of 70kDa heat shock protein(HSP70) in C6 glioma cells.
Methods : The C6 glioma cells belong to control group were incubated with DMEM culture solution, the cells belong to dexamethasone group were incubated with dexamethasone for 6 hours, and the cells belong to fluoxetine group were incubated with fluoxetine for 1, 6, 24, and 72 hours, separately, and then exposed to dexamethasone for an additional 6 hours. Crude extracts from control, dexamethasone and fluoxetine-treated C6 glioma cells were separated on a 10% SDS-PAGE and probed with anti-HSP70mAb.
Results : 1) Dexamethasone(10uM, 6hours) reduced the level of HSP70 expression relative to control, but this reduction was not statistically significant. 2) Pretreatment with fluoxetine(10uM, 1, 6, 24, and 72 hours) and exposure to dexamethasone(10uM, 6hours) decreased the level of HSP70 expression according to the duration of fluoxetine treatment. 3) Fluoxetine significantly reduced the level of HSP70 at 24 and 72 hours compared to control. However, compare to the level of HSP70 expression at 24 hours, the level of HSP70 expression at 72 hours was elevated.
Conclusions : These findings suggest that dexamethasone and fluoxetine may affect HSP70 expression through effects on GR.

국문 초록 (Abstract)

목 적：시상하부-뇌하수체-부신(hypothalamic-pi -tuitary-adrenal；HPA) 축의 과다활동은 우울 장애의 특징이며, 항우울제 치료는 HPA 축의 과다활동을 정상화시킨다. 또한 스트레스에 반응하여 열충격 단백질(heat shock protein；HSP)이라고 알려진 일군의 단백질이 증가되는데, HSP의 일종인 70kDa HSP(HSP70)는 신경세포의 스트레스를 조기에 알려주는 민감한 표식자이며, glucocorticoid 수용체(glucocorticoid receptor；GR)에 결합하여 활성화시킨다고 알려져 있다. 그러므로 본 연구에서는 glucocorticoid, fluoxetine, 그리고 HSP70의 관련성을 규명하고자 하였다. 방 법：흰쥐 C6 glioma 세포에 glucocorticoid계의 일종인 dexamethasone을 6시간 동안 처리했을 때 HSP70의 발현 정도를 면역탁본법(im unoblot)을 이용해 조사하여 glucocorticoid가 신경세포에서 HSP70의 발현에 미치는 효과를 검증하였다. 또한 fluoxetine을 1, 6, 24, 72시간 동안 전처리한 이후 dexamethasone에 노출시켜서 HSP70의 발현 양상을 비교 조사하였다. 결 과：첫째, dexamethasone이 HSP70의 발현을 감소시키는 경향이 나타났다. 둘째, fluoxetine으로 전처리이준석 등 73 한 이후 dexamethasone에 노출시켰을 때 HSP70의 발현이 전처리 기간에 따라 감소되었으며, 특히 24시간 및 72시간에서는 대조군에 비하여 의미 있게 감소되었다. 셋째, fluoxetine을 전처리한 이후 24시간까지는 HSP70의 발현이 지속적으로 감소되었지만 72시간에서는 미약하지만 다시 증가되는 경향이 나타났다.
결 론：본 연구 결과는 fluoxetine 치료가 단기적으로 GR을 전위시키는 기전을 통하여 GR 저항성을 개선시킴으로써 HPA 축의 과다활동 및 혈청 cortisol 농도의 상승을 정상화시킬 뿐만 아니라 HSP70의 발현을 낮추고, 더 나아가 주요 우울증의 증상을 개선시킬 것이라는 새로운 항우울 작용을 제시한다.

참고문헌 (Reference)

1 "cAMP-dependent phosphorylation of soluble and crude microtubule fraction of rat cerebral cortex after prolonged desmethylimipramine treatment" 172 : 305-316, 1989

2 "cAMP response element-mediated gene transcription is upregulated by chronic antidepressant treatment" 20 : 4030-4036, 2000

3 "Type II glucocorticoid receptors are expressed in oligodendrocytes and astrocytes" 27 : 360-373, 1990

4 "Transcriptional regulation of heat shock genes" 267 : 21987-21990, 1992

5 Pratt WB, "The role of heat shock proteins in regulating the function,folding,and trafficking of the glucocorticoid receptor" 268 : 21455-21458, 1993

6 "The effects of cortisol on heat shock protein 70 levels in two fish species" 124 : 97-105, 2001

7 "The antidepressant fluoxetine,idazoxan and phenelzine alter corticotropin-releasing hormone and tyrosine hydroxylase mRNA levels in rat brain:therapeutic implications" 572 : 117-125, 1992

8 "Stress-induced heat shock protein 70 expression in adrenal cortex:an adrenocorticotrophic hormone-sensitive,age-dependent response" 88 : 9873-9877, 1991

9 "Stress,glucocorticoids,and damage to the nervous system:the current state of confusion" 1 : 1-19, 1996b

10 "Steroids and depression" 38 : 537-559, 1991

11 "Steroid-induced psychiatric syndromes" 5 : 319-332, 1983

12 "Steroid-independent translocation of the glucocorticoid receptor by the antidepressant desipramine" 52 : 571-581, 1997

13 "Steroid receptor interactions with heat shock protein and immunophilins chaperones" 18 : 306-360, 1997

14 "Short-term administration of fluoxetine and venlafaxine decreases corticosteroid receptor mRNA expression in the rat hippocampus" 306 : 16-, 2001

15 "Serotonin regulates type II corticosteroid receptor binding in hippocampal cell cultures" 10 : 1745-1752, 1990

16 "Serotonin 5-HT2A receptor activation inhibits inducible nitric oxide synthase activity in C6 glioma cells" 61 : 1819-1827, 1997

17 "Role of selective serotonin reuptake inhibitors in psychiatric disorders:a comprehensive review" 27 : 85-102, 2003

18 "Pulse-dosing and conventional application of doxepin:effects on psychopathology and hypothalamus-pituitary-adrenal axis" 17 : 156-160, 1997

19 "Prozac(fluoxetine, Lilly 110140) the first selective serotonin uptake inhibitor and antidepressant drug: Twenty years since its first publication" 57 : 411-441, 1995

20 "Pituitary-adrenal system regulation and psychopathology during amitriptyline treatment in elderly patients and normal comparison subjects" 153 : 93-99, 1996

21 "Maturation of steroid receptors:an example of functional cooperation among molecular chaperones and their associated proteins" 5 : 76-86, 2000

22 "Long-term treatment with antidepressants increases glucocorticoid receptor binding and gene expression in cultured rat hippocampal neurons" 11 : 887-895, 1999

23 "How cells respond to stress" 268 : 56-64, 1993

24 "Hippocampal formation volume,memory dysfunction,and cortisol levels in patients with Cushing's disease" 32 : 756-765, 1992

25 "Heat-shock protein protection" 22 : 97-99, 1999

26 "Heat shock proteins:endogenous modulators of apoptotic cell death" 286 : 433-442, 2001

27 "Heat shock proteins and molecular chaperones:mediators of protein conformation and turnover in the cell" 78 : 365-, 1994

28 "Heat shock protein expression in vulnerable cells of the rat hippocampus as an indicator of excitation-induced neuronal stress" 12 : 3004-3009, 1992

29 "Glucocorticoids regulate the synthesis of HSP27 in rat brain slices" 847 : 9-17, 1999

30 "Glucocorticoids inhibit glucose transport in cultured hippocampal neurons and glia" 52 : 57-62, 1990

31 "Glucocorticoid receptors in major depression:Relevance to pathophysiology and treatment" 49 : 391-404, 2001

32 "Fluoxetine:a selective inhibitor of serotonin uptake" 11 : 17-34, 1991

33 Tomitaka M, "Fluoxetine prevents PCP-and MK801-induced HSP70 expression in injured limbic cortical neurons of rats" 47 : 836-841, 2000

34 "Etiologic implications of corticosteroid changes in affective disorder" 3 : 135-155, 1985

35 "Effects of GH,prolactin and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba" 161 : 413-421, 1999

36 "Early clinical response in depression to venlafaxine hydrochloride" 29 : 630-, 1991

37 "Differential regulation by dexamethasone of glucocorticoid receptor messenger RNA concentrations in neuronal cultures derived from fetal rat hypothalamus and cerebral cortex" 10 : 227-235, 1990

38 "Differential cellular distribution and dynamics of HSP70,cyclooxygenase-2,and c-Fos in the rat brain after transient focal ischemia or kainic acid" 80 : 221-232, 1997

39 "Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function" 261 : 4909-4914, 1986

40 "Cortisol modulates HSP90 mRNA expression in primary cultures of trout hepatocytes" 129 : 679-685, 2001

41 "Corticosterone enhances kainic acid-induced calcium elevation in cultured hippocampal neurons" 59 : 1033-1040, 1992

42 "Clinical correlates of major depression in Cushing's disease" 31 : 302-306, 1998

43 "Chronic treatment of C6 glioma cells with antidepressant drugs increases functional coupling between a G protein(Gs)and adenylyl cyclase" 64 : 724-732, 1995

44 "Chronic glucocorticoid receptor activation impairs CREB transcriptional activity in clonal neurons" 304 : 720-723, 2003

45 Nestler EJ, "Chronic antidepressant alter the subcellular distribution of cyclic AMP-dependent protein kinase in rat frontal cortex" 53 : 1644-1647, 1989

46 "Cell specific expression of Hsp70 in neurons and glia of the rat hippocampus after hyperthermia and kainic acid-induced seizure activity" 71 : 265-278, 1999

47 "Antidepressants increase glucocorticoid and mineralocorticoid receptor mRNA expression in the rat hippocampus in vivo" 55 : 621-626, 1992

48 "Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes" 232 : 522-524, 1986