Targeted Gq-GPCR activation drives ER-dependent calcium oscillations in chondrocytes

Ryan C McDonough; Rachel M Gilbert; Jason P Gleghorn; Christopher Price

doi:10.1016/j.ceca.2021.102363

Targeted Gq-GPCR activation drives ER-dependent calcium oscillations in chondrocytes

Cell Calcium. 2021 Mar:94:102363. doi: 10.1016/j.ceca.2021.102363. Epub 2021 Jan 30.

Authors

Ryan C McDonough¹, Rachel M Gilbert², Jason P Gleghorn³, Christopher Price⁴

Affiliations

¹ Department of Biomedical Engineering, University of Delaware, United States. Electronic address: ryancmcd@udel.edu.
² Department of Biomedical Engineering, University of Delaware, United States. Electronic address: rgilbert@udel.edu.
³ Department of Biomedical Engineering, University of Delaware, United States. Electronic address: gleghorn@udel.edu.
⁴ Department of Biomedical Engineering, University of Delaware, United States. Electronic address: cprice@udel.edu.

PMID: 33550208
DOI: 10.1016/j.ceca.2021.102363

Abstract

The temporal dynamics of calcium signaling are critical regulators of chondrocyte homeostasis and chondrogenesis. Calcium oscillations regulate differentiation and anabolic processes in chondrocytes and their precursors. Attempts to control chondrocyte calcium signaling have been achieved through mechanical perturbations and synthetic ion channel modulators. However, such stimuli can lack both local and global specificity and precision when evoking calcium signals. Synthetic signaling platforms can more precisely and selectively activate calcium signaling, enabling improved dissection of the roles of intracellular calcium ([Ca²⁺]_i) in chondrocyte behavior. One such platform is hM3Dq, a chemogenetic DREADD (Designer Receptors Exclusively Activated by Designer Drugs) that activates calcium signaling via the Gα_q-PLCβ-IP₃-ER pathway upon administration of clozapine N-oxide (CNO). We previously described the first-use of hM3Dq to precisely mediate targeted, synthetic calcium signals in chondrocyte-like ATDC5 cells. Here, we generated stably expressing hM3Dq-ATDC5 cells to investigate the dynamics of Gα_q-GPCR calcium signaling in depth. CNO drove robust calcium responses in a temperature- and concentration-dependent (1 pM-100 μM) manner and elicited elevated levels of oscillatory calcium signaling above 10 nM. hM3Dq-mediated calcium oscillations in ATDC5 cells were reliant on ER calcium stores for both initiation and sustenance, and the downregulation and recovery dynamics of hM3Dq after CNO stimulation align with traditionally reported GPCR recycling kinetics. This study successfully generated a stable hM3Dq cell line to precisely drive Gα_q-GPCR-mediated and ER-dependent oscillatory calcium signaling in ATDC5 cells and established a novel tool to elucidate the role that GPCR-mediated calcium signaling plays in chondrocyte biology, cartilage pathology, and cartilage tissue engineering.

Keywords: Calcium signaling; Chemogenetics; Chondrocyte; Designer receptors exclusively activated by Designer drugs; G protein coupled receptor activation.

MeSH terms

Calcium Signaling* / drug effects
Cell Line
Chondrocytes / drug effects
Chondrocytes / metabolism
Clozapine / analogs & derivatives
Clozapine / pharmacology
Endoplasmic Reticulum / drug effects
Endoplasmic Reticulum / metabolism*
GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism*
Humans
Ion Channel Gating / drug effects
Receptors, G-Protein-Coupled / metabolism*
Time Factors

Substances

Receptors, G-Protein-Coupled
GTP-Binding Protein alpha Subunits, Gq-G11
Clozapine
clozapine N-oxide