Effect of hippocalcin on the differentiation of hippocampal neural precursor cells

Abstract

Hippocalcin (Hpca) is a neuronal calcium sensor protein expressed in mammalian brain. However, its function in neural stem cell differentiation has not been studied yet. Here, we investigated the effect of Hpca on the differentiation at rat embryonic hippocampal developmental stages. We performed microarray using isolated RNA from E16, E17 and E18 hippocampal neural precursor cells (HNPCs). We found that significant differences in gene expression were observed between E16 and E17 but not between E17 and E18. Surprisingly, when we overexpressed Hpca in E16 HNPCs, expression levels of nerve-growth factors such as NT-3, NT-4/5, BDNF, together with proneural basic helix loop helix (bHLH) transcription factors NeuroD and neurogenin 1 (ngn1) were significantly increased in the presence of bFGF, but not in E17 HNPCs. To confirm the reason why Hpca has a different role between E16 and E17, we transfected Hpca siRNA into E16 or E17 HNPCs. Interestingly, expression levels of NT-3, NT-4/5, BDNF, NeuroD and ngn1 were decreased by knockdown of Hpca in E16, while they were increased in E17. Moreover, overexpression of Hpca increased expression level of glial fibrillary acidic protein (GFAP), an astrocyte marker, in E16 HNPCs, but not in E17 HNPCs. Furthermore, we showed that STAT3 (Ser727) activation is involved in Hpca-mediated astrocyte differentiation. We can conclude for the first time that Hpca has an opposite role in expression of neurotrophins and proneural bHLH transcription factors between E16 and E17 HNPCs, and it acts as critical regulator of astrocyte differentiation in E16 HNPCs through STAT3 activation.|Hippocalcin 은 포유류의 신경조직에만 특이적으로 발현하는 칼슘결합단백질이다. 그러나, hippocalcin을 통한 해마신경전구세포 [hippocampal neural precursor cells (HNPCs)]의 분화조절 메커니즘은 아직까지 보고되어지지 않고 있다. 본 연구에서는 hippocalcin이 해마 발달 단계에 따른 신경 분화 조절에 미치는 영향을 규명하고자 하였다. Microarray 분석을 통하여 embryonic day (E) 16과 E17 해마신경전구세포 사이에 상당한 유전자 발현의 차이를 관찰하였다. 놀랍게도, E16에서는 hippocalcin 과발현에 의해 NT-3, NT-4/5, BDNF와 같은 neurotopbins와 proneural bHLH transcription factors인 NeuroD와 ngn1의 발현이 증가하였고, 이와 반대로 E17 에서는 이들의 발현이 감소하는 현상을 확인하였다. RNA간섭방법을 이용하여 hippocalcin의 발현을 저해시켰을 때에는 neurotrophins와 proneural bHLH transcription factors 발현이 E16에서는 감소하였고, E17에서는 증가하였다. 또한 E16에서는 hippocalcin 과발현에 의해 astrocyte 분화가 촉진되었고, E17에서는 영향이 없었다. 이와 같은 결과는 hippocalcin이 해마 발달단계에 따라 분화조절이 반대로 일어나는 것을 암시한다. Immunoblot 결과는 이러한 분화조절이 STAT3 (Ser727) 인산화에 의해서 조절되는 것을 보여주었다. Hippocalcin이 과발현된 E16 세포에 Stat3 억제제인 S3I-201이나 Stat3 siRNA를 처리하였을 때, 신경성장인자들과 GFAP의 발현이 차단되었다. 이러한 결과들은 hippocalcin이 신경교세포의 dendrite 길이의 증가뿐 아니라 STAT3와 신경성장인자들을 경유하는 경로를 통해 해마신경세포 분화조절에 있어서 중요하게 작용한다고 사료되어진다.; Hippocalcin (Hpca) is a neuronal calcium sensor protein expressed in mammalian brain. However, its function in neural stem cell differentiation has not been studied yet. Here, we investigated the effect of Hpca on the differentiation at rat embryonic hippocampal developmental stages. We performed microarray using isolated RNA from E16, E17 and E18 hippocampal neural precursor cells (HNPCs). We found that significant differences in gene expression were observed between E16 and E17 but not between E17 and E18. Surprisingly, when we overexpressed Hpca in E16 HNPCs, expression levels of nerve-growth factors such as NT-3, NT-4/5, BDNF, together with proneural basic helix loop helix (bHLH) transcription factors NeuroD and neurogenin 1 (ngn1) were significantly increased in the presence of bFGF, but not in E17 HNPCs. To confirm the reason why Hpca has a different role between E16 and E17, we transfected Hpca siRNA into E16 or E17 HNPCs. Interestingly, expression levels of NT-3, NT-4/5, BDNF, NeuroD and ngn1 were decreased by knockdown of Hpca in E16, while they were increased in E17. Moreover, overexpression of Hpca increased expression level of glial fibrillary acidic protein (GFAP), an astrocyte marker, in E16 HNPCs, but not in E17 HNPCs. Furthermore, we showed that STAT3 (Ser727) activation is involved in Hpca-mediated astrocyte differentiation. We can conclude for the first time that Hpca has an opposite role in expression of neurotrophins and proneural bHLH transcription factors between E16 and E17 HNPCs, and it acts as critical regulator of astrocyte differentiation in E16 HNPCs through STAT3 activation.