CDK5-induced p-PPARγ(Ser 112) downregulates GFAP via PPREs in developing rat brain: effect of metal mixture and troglitazone in astrocytes

P Singh,S Tripathi,P Srivastava,S Bandyopadhyay,S Sanyal,A Rai,R Kushwaha

doi:10.1038/cddis.2013.514

Abstract

The peroxisome proliferator-activated receptor gamma (PPARγ), a group of ligand-activated transcriptional factors, is expressed in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Here, we investigated the role of PPARγ in regulating GFAP using a mixture of As, Cd and Pb (metal mixture, MM) that induces apoptosis and aberrant morphology in rat brain astrocytes. We observed a phospho PPARγ (serine 112 (S112)) (p-PPARγ (S112))-mediated downregulation of GFAP in the MM-exposed astrocytes. We validated this using pure PPARγ agonist, troglitazone (TZ). As reported with MM, TZ induced astrocyte damage owing to reduced GFAP. In silico analysis in the non-coding region of GFAP gene revealed two PPARγ response elements (PPREs); inverted repeat 10 and direct repeat 1 sequences. Gel shift and chromatin immunoprecipitation assays demonstrated enhancement in binding of p-PPARγ (S112) to the sequences, and luciferase reporter assay revealed strong repression of GFAP via PPREs, in response to both MM and TZ. This indicated that suppression in GFAP indeed occurs through direct regulation of these elements by p-PPARγ (S112). Signaling studies proved that MM, as well as TZ, activated the cyclin-dependent kinase 5 (CDK5) and enhanced its interaction with PPARγ resulting into increased p-PPARγ (S112). The p-CDK5 levels were dependent on proximal activation of extracellular signal-regulated protein kinase 1/2 and downstream Jun N-terminal kinase. Taken together, these results are the first to delineate downregulation of GFAP through genomic and non-genomic signaling of PPARγ. It also brings forth a resemblance of TZ with MM in terms of astrocyte disarray in developing brain.

Highlights

Glial fibrillary acidic protein (GFAP) preserves shape and arbor of astrocyte processes contributing to mechanical strength of the cell.[1]
We investigated the phosphorylation of PPARg at serine 112 (S112) residue, and probed binding of PPARg (S112) to functional peroxisome proliferator-activated receptor gamma (PPARc) response elements (PPREs) in GFAP gene in metal mixture (MM)- and TZ-exposed astrocytes
T0070907 prevented the rise in Phospho-peroxisome proliferator-activated receptor gamma (p-PPARg) (S112) (Figure 1d) and fall in GFAP (Figure 1e)

Summary

Introduction

GFAP preserves shape and arbor of astrocyte processes contributing to mechanical strength of the cell.[1]. Group of ligand-activated transcription factors influence metabolism and functions of astrocytes in brain.[9,10] The PPAR gamma (PPARg) promotes apoptosis[11] and participates in differentiation of astrocytes.[12] the role of PPARg in GFAP regulation is unexplored. Activation of cyclin-dependent kinase 5 (CDK5) is reported to increase phosphorylation of PPARg in pancreatic b-cells,[18] human embryonic kidney cells,[19] colon cancer cells[20] and adipose tissue.[21] The CDK5 and mitogen-activated protein kinases (MAPKs) are associated with cytoskeletal proteins in rat brain,[22] and cross talk between CDK5 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) moderates neuroplasticity[23,24] and regulates glial migration.[25] the interdependence of these kinases in regulating GFAP is unreported. We reported earlier that a mixture of As, Cd and Pb (metal mixture (MM)), at doses relevant to human exposure, attenuated GFAP levels, adversely affected astrocyte morphology and induced apoptosis in developing rat brain.[26]

Methods

Results

Conclusion