Overexpression of pitx3 upregulates expression of BDNF and GDNF in SH‐SY5Y cells and primary ventral mesencephalic cultures

Abstract

The transcription factor Pitx3 plays an important role in the development of midbrain to promote the growth and differentiation of dopamine neurons. The present study has demonstrated that overexpression of Pitx3 in SH-SY5Y cells and primary ventral mesencephalic (VM) cultures significantly increased the mRNA levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), and remarkably elevated the protein levels of these two neurotrophic factors. Our data provide the first evidence that pitx3-expressing cells are able to upregulate the expression of BDNF and GDNF. Therefore, Pitx3 might be a good target for the treatment of Parkinson's disease.

Driving GDNF expression: The green and the red traffic lights

“…Very recently, it was described that the overexpression of Pitx3 in SH-SY5Y cells and primary ventral mesencephalic cultures significantly increases GDNF and BDNF mRNA levels and elevates their protein levels (Peng et al, 2007). Since Pitx3 is a transcription factor with an important role in the growth and differentiation of dopaminergic neurons (reviewed by Smidt et al, 2004) this finding may represent the very first step, as far as we are aware, for unravelling how GDNF expression is regulated during the development of the nigrostriatal system.…”

Pitx3 Is a Critical Mediator of GDNF-Induced BDNF Expression in Nigrostriatal Dopaminergic Neurons

“…For GDNF treatments, E14 rat primary VM cells were treated after 7 days in vitro (DIV) with 10 ng/ml recombinant rat GDNF (Sigma) in 0.9% saline or with 0.9% saline alone (control) for 3, 24 or 48 h. In some cases, 10 g/ml NF-B inhibitor (SN50; Calbiochem) were added for 1 h to the culture medium before the GDNF treatment. (Peng et al, 2007). Under these conditions, one Pitx3-specific shRNA oligonucleotide (antisense: 5Ј-GCACGCCUCU-UUCAGCUAUdTdT-3Ј; sense: 5Ј-AUAGCUGAAAGAGGCGUGCdTdT-3Ј; targeting sequences around position 956 to 974 in Pitx3 exon 4, GenBank accession no.. NM_008852.4) resulted in ϳ80% knock-down of Pitx3 mRNA (data not shown).…”

“…Immunostaining of cultured cells, paraffin sections and free-floating cryosections (40 m) was performed as described previously (Brodski et al, 2003;Peng et al, 2007). Polyclonal rabbit antisera were directed against NF-B (1:100; Cell Signaling Technology), BDNF (1:500 (Sc-546); Santa Cruz Biotechnology), Pitx3 (1: 300; Zymed), and cleaved (activated) Caspase-3 (cCasp3, 1:100; Cell Signaling Technology).…”

“…SN50 (10 g/ml) was added 30 min before 10 ng/ml or 20 ng/ml GDNF was given to the medium. HEK293 cells were cotransfected with 200 ng/well pGL3-BDNF wild-type or mutant promoter vector, 400 ng/ well pcDNA3.1-Pitx3 (Peng et al, 2007) or pcDNA3.1 alone, and 8 ng/ well pRL-SV40 using Lipofectamine LTX and Plus Reagent (Invitrogen). Cells were lysed in Passive Lysis Buffer 24 h (BDNF promoter assays) or 36 h (Pitx3 promoter assays) post-transfection, and Firefly and Renilla Luciferase luminescence were measured in a Centro LB 960 luminometer (Berthold Technologies) using the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturers' instructions.…”

Microcapsule Co-Culture System Enhances Neural Differentiation of Mesenchymal Stem Cells

“…at gene or protein level 24) . More importantly, SHSY5Y cells can secrete some soluble factors, including glial cell line-derived neurotrophic factor (GDNF) 25) , brain-derived neurotrophic factor (BDNF) 26) or IGF-1 27) , etc., and some of these factors are reported to enhance the differentiation of MSCs into neural lineages 28) . Because the molecular weight of above factors is below 20 KDa, which allows the soluble factors to diffuse through the membranes of transwell or microcapsule (molecular weight cutoff 150KDa) freely to further enhance the differentiation of MSCs into neural progenitors.…”