Protective effect of JAK3 inhibition on growth factor deprivation induced neural progenitor cell death

Apoptosis is evolutionally conserved, and plays central roles during normal development of the nervous system. The number of embryonic neuroepithelial cells (NECs) is regulated by programmed cell death when NECs are substantially proliferated by growth factors(GFs). The extrinsic factors such as epidermal growth factor (EGF), basic fibroblast growth factor(bFGF), as well as intrinsic protein regulate growth, proliferation and differentiation. Therefore, in culture system, addition of both bFGF and EGF is critical to maintain and expand NECs. The withdrawal of the seGFs induced apoptosis through intrinsic mitochondrial pathway in NECs. However, the mechanisms underlying the fate of NECs to either survive or dieare incompletely understood. In the brain, a specific JAK3 inhibitor aids in the survival of a transgenic amyotrophic lateral sclerosis mouse model. We previously reported that JAK3 inhibition induced neuronal differentiation accompanied by neurite outgrowth in enriched condition of GFs such as EGF, bFGF. In the present study, effect of JAK3 inhibition was examined on GFs withdrawal induced death of cultured NECs from embryonicmice brain (E13.5 days). The GFs withdrawal decreased cell vibility in time-dependent manner, however treatment of JAK3 inhibitor (WHI-p131) increased cellviability. The try pan bluestained cells, which were dead, were increased in GFs-withdrawal condition, incontrast those were decreased by JAK3 inhibitor. For further confirmation, we also performed propidiumiodide (PI) and calcein-AM double staining. The number of calcein-AM stained cells, which were alive, was increased strikingly by JAK3 inhibitor. However, the number of PI-positive cells, which were dead, was similar between JAK3 inhibitor treated and non-treated condition. To confirmidentity of survival cells, the cells were stained with immunocytochemical markers for several cell types. JAK3 inhibitor increased the number of nestin, SOX2 (markers of NPCs) and MAP2(a marker of neuron) positive cells. But capability of proliferation of NSCs was not affected by JAK3 inhibitor. GFs withdrawal is known to induce apoptosis, which could detect by TUNEL(terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nickendlabeling)assay. The number of TUNEL positive cells was decreased in a JAK3 inhibitor dose dependently. And we confirmed casapse-3 activity, since caspase-3 is a caspase protein that plays a central role in the execution of cell apoptosis. Caspase-3 activity that was not affected by JAK3 inhibitor was decreased by caspase-3 inhibitor, but cell viability was not changed by caspase-3 inhibitors. In GFs withdrawal condition, phosphorylation of JAK3 was increased but treatment of JAK3 inhibitor reduced phosphorylation of JAK3. where sGFs withdrawal resulted in decrement of phosphorylation of AKT, ERK and was increment pattern of phosphorylation of JNK, JAK3 inhibitors shown a tendency to increase phosphorylation of AKT and more decreased phosphorylation of ERK, JNK than non treated group. And, JAK3 inhibitor shown a tendency to decrease of Bim-EL protein level, which is BH3-only protein of pro-apoptotic bcl-2 family. And, expression of adhesion molecules such as N-cadherin, NCAM was increased by JAK3 inhibitor. In summary, we identified that GF withdrawal induced apoptosis of NSCs, but inhibition of JAK3 drives NSCs toward cell survival. Also, inhibition of JAK3 promotes NSCs to differentiate into mature neuron accompanied by neurite outgrowth via increase of neuronal adhesion molecule expression such as N-cadherin, NCAM. Therefore, we propose that JAK3 may have acritical role on not only survival but also differentiation of NSCs.

Author dc:creator

• 임, 소연

Contributors dc:contributor

• 백, 은주
• 대학원 의생명과학과
• 201224137

• 신경상피세포
• 성장인자
• JAK3
• 세포생존
• 세포사멸
• 신경발생
• neuroepithelialcells (NESc)
• growthfactors (GFs)
• cellsurvival
• celldeath
• neurogenesis

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000015975
000000015975

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/10852