Roles of p38 MAPK Inhibitors and Ca²^(+) in Primary Astroglia Insulted to Death by Zinc

Abstract

Zinc is a necessary catalytic or structural factor of various proteins. It acts as a neuromodulator that is released by neural activity at a number of central excitatory synapses and regulate synaptic function. However, in pathological condition, it can be released in excess, and excess zinc in the extracellular fluid acts as a neurotoxin in the brain. Several studies have indicated that zinc has a role in neuronal death related to transient global ischemia, seizures, and other neurological diseases. Molecular mechanisms underlying zinc-induced cell death of brain cells have been explored using primary cortical neuron or glia cultures in vitro. Chronic treatment of cortical neurons and astroglia in culture with zinc 35 uM causes to cell death. Astroglial cells insulted by zinc elongated in shape, swelled and rapidly detached from culture plate. The rapid change and characteristic loss of morphological integrity of astroglial cells appear to proceed prior to cell death. The present study was aimed to investigate the mechanism underlying the rapid change and characteristic loss of morphological integrity of zinc-treated astroglia. The MAP kinase (MAPK) pathway controls various cellular functions such as cell proliferation, cell differentiation and apoptosis. The MAPK family consists of ERK1/2, JNK/SAPK, and p38. In the present study, it was examined whether MAPK pathways were involved in zinc-induced astroglial cell death. It was found that p38 and JNK were strongly activated in zinc-treated astroglia. To examine the roles of p38 and JNK MAPK in zinc-treated astroglia, the p38 MAPK inhibitors, SB202190 and SB 203580, and the JNK inhibitor, SP600125, were added in the culture medium of zinc-treated astroglia. Although p38 and JNK inhibitors did not protect zinc-induced cell death, the p38 inhibitors (SB203580 and SB202190), but not the JNK inhibitor (SP600125), protected cell morphology, keeping zinc-treated astroglia attached on the culture plate. The distinct morphology protection by p38 inhibitors was accompanied with enhanced actin-ring formation around cell membrane. Other protein kinase inhibitors (MEK1, JNK, PKC, PKA inhibitor or CAMK inhibitor), phosphatase inhibitor, caspase inhibitor, and PARP inhibitor did not protect cell morphology. Most inhibitors of ROS- generating enzyme did not protect cell morphology. Similar to that by p38 inhibitors, the Ca²^(+) ionophore, A23187, completely protected cell morphology, but not cell death of zinc-treated astroglia. However, the abnormal enhancement of actin-ring formation around cell membrane and activation produced by p38 MAPK inhibitors was not produced by A23187. To investigate the mechanism of morphology protection by p38 MAPK inhibitors, altered protein profiles in control astroglia vs. zinc-treated astroglia vs. zinc- and SB203580-treated astroglia were analzed using two-dimensional SDS PAGE analysis followed by MALDI-TOF analysis. The resulting data showed that calrecticurin and pea 15 protein, were down- and up-regulated, respectively, after Zn^(+)² treatment, and these altered protein levels were reversed by SB203580. Calreticulin is a cellular factor that regulates maintenance of intracellular Ca2+ homeostasis and cell adhesion. Pea-15 regulates signaling pathways which control cell proliferation and cell death. The results of the present study suggest that p38 MAPK-regulated and/or Ca²^(+)-mediated cellular mechanisms are important cellular factors involved in cell morphology regulation in astroglia that are challenged to cell death by zinc.;뇌에는 적절한 양의 아연 이온이 존재하며 시냅스 전달의 역할을 한다. 그러나 병리학적인 상황에서 과도하게 방출된 아연은 뇌 세포의 사멸을 유도한다. 배양 중인 마우스의 대뇌 피질 성상교세포에 35uM의 아연을 처리했을 때 시간이 지남에 따라 세포 사멸이 진행되고 24시간 후 세포 배양 접시 바닥에 붙어 있던 세포들이 위로 둥둥 떠오르게 된다. 이러한 아연에 의한 세포 사멸 과정 중에 p38과 JNK가 활성화 됨을 확인하였다. 아연과 JNK 억제제(SP600125)를 함께 처리했을 때 세포 사멸이 유도되고 세포 모양이 망가짐을 관찰하였으나 아연과 p38 억제제(SB203580)를 함께 처리했을 때는 사멸이 일어나기는 하지만 세포 모양이 유지되어 세포가 바닥에 잘 붙어 있음을 발견했다. 또한, 아연과 P38 억제제 (SB203580)를 함께 처리했을 때 세포 모양이 유지되고 세포막 주변에 엑틴이 고리 모양으로 필라멘트 형상이 강화되는 현상을 관찰했다. 아연에 의한 세포 사멸 과정에 활성산소종이 관여한다는 보고가 있어 여러 종류의 항산화제(NS398, DPI, Allopurinol, PBN, NAME or PDTC)를 처리해 본 결과 세포 모양이 보호되는 것을 볼 수 없었다. 사멸 신호 전달 과정에 중요한 역할을 하는 caspase 억제제(zVAD)나 PARP 억제제(Benzimide)에 의해서도 세포 모양이 보호되지 않았다. 칼슘이온을 투과시키는 역할을 하는 A23187과 아연을 함께 처리했을 때 사멸은 일어나나 세포 모양이 유지되어 p38 억제제를 아연과 함께 처리했을 때와 같은 효과를 확인할 수 있었으며 이에 근거해 세포 모양 유지에 칼슘이 관여함을 알 수 있었다. 아연과 p38 억제제를 함께 처리한 후 2차 전기 영동을 통해 단백질 분석을 했을 때 careticulin이 변화했음을 관찰했다. 이러한 결과는 careticulin이 아연을 처리한 성상교세포의 모양 보호에 중요한 역할을 할 것이라는 것을 시사한다.