- Authors: Kobayashi Y. et al.
- Year: 2021
- Journal: FASEB Bioadv 3 744-767
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: hTERT RPE-1
Description: hTERT-immortalised human retinal pigment epithelial cells
MCHR1:EGFP clone cells were treated with PDLIM5 siRNA#1 and #2 together, and then the rescue HA-PDLIM5 vector or mock vector was transiently transfected with jetOPTIMUS. PDLIM5-/- hTERT-RPE1 cells were co-transfected with HA-tagged PDLIM5 and MCHR1:EGFP by jetOPTIMUS.
The primary cilium is a plasma membrane-protruding sensory organelle that efficiently conveys signaling cascades in a highly ordered microenvironment. Its signaling is mediated, in part, by a limited set of GPCRs preferentially enriched in the cilium membrane. This includes melanin-concentrating hormone (MCH) receptor 1 (MCHR1), which plays a role in feeding and mood. In addition to its receptor composition, the length of the cilium is a characteristic parameter that is implicated in its function. We previously found that MCH can dynamically shorten cilia length via the Gi/o and Akt pathways in both MCHR1-expressing hTERT-RPE1 cells (hRPE1 cells) and rat hippocampal neurons. However, the detailed mechanisms by which MCH regulates cilia length through ciliary MCHR1 remains unclear. In this study, we aimed to determine the transcriptome changes in MCHR1-expressing hRPE1 cells in response to MCH to identify the target molecules involved in cilia length regulation via MCHR1 activation. RNA sequencing analysis of ciliated cells subjected to MCH treatment showed upregulation of 424 genes and downregulation of 112 genes compared with static control cells. Validation by quantitative real-time PCR, knocking down, and CRISPR/Cas9-mediated knockout technology identified a molecule, PDZ and LIM domain-containing protein 5 (PDLIM5). Thus, it was considered as the most significant key factor for MCHR1-mediated shortening of cilia length. Additional analyses revealed that the actin-binding protein alpha-actinin 1/4 is a crucial downstream target of the PDLIM5 signaling pathway that exerts an effect on MCHR1-induced cilia shortening. In the endogenous MCHR1-expressing hippocampus, transcriptional upregulation of PDLIM5 and actinin 1/4, following the application of MCH, was detected when the MCHR1-positive cilia were shortened. Together, our transcriptome study based on ciliary MCHR1 function uncovered a novel and important regulatory step underlying cilia length control. These results will potentially serve as a basis for understanding the mechanism underlying the development of obesity and mood disorders.