Targeted Activation of G-Protein Coupled Receptor-Mediated Ca²⁺ Signaling Drives Enhanced Cartilage-Like Matrix...

Intracellular calcium ([Ca²⁺]_i) signaling is a critical regulator of chondrogenesis, chondrocyte differentiation, and cartilage development. Calcium (Ca²⁺) signaling is known to direct processes that govern chondrocyte gene expression, protein synthesis, cytoskeletal remodeling, and cell fate. Control of chondrocyte/chondroprogenitor Ca²⁺ signaling has been attempted through mechanical and/or pharmacological activation of endogenous Ca²⁺ signaling transducers; however, such approaches can lack specificity and/or precision regarding Ca²⁺ activation mechanisms. Synthetic signaling platforms permitting precise and selective Ca²⁺ signal transduction can improve dissection of the roles that [Ca²⁺]_i signaling plays in chondrocyte behavior. One such platform is the chemogenetic DREADD (designer receptor exclusively activated by designer drugs) hM3Dq, which activates [Ca²⁺]_i signaling via the Gα_q-PLCβ-IP₃-ER pathway upon clozapine N-oxide (CNO) administration. We previously demonstrated hM3Dq's ability to precisely and synthetically initiate robust [Ca²⁺]_i transients and oscillatory [Ca²⁺]_i signaling in chondrocyte-like ATDC5 cells. Here, we investigate the effects that long-term CNO stimulatory culture have on hM3Dq [Ca²⁺]_i signaling dynamics, proliferation, and protein deposition in 2D ATDC5 cultures. Long-term culturing under repeated CNO stimulation modified the temporal dynamics of hM3Dq [Ca²⁺]_i signaling, increased cell proliferation, and enhanced matrix production in a CNO dose- and frequency-dependent manner, and triggered the formation of cell condensations that developed aligned, anisotropic neotissue structures rich in cartilaginous proteoglycans and collagens, all in the absence of differentiation inducers. This study demonstrated Gα_q-G-protein coupled receptor (GPCR)-mediated [Ca²⁺]_i signaling involvement in chondroprogenitor proliferation and cartilage-like matrix production, and it established hM3Dq as a powerful tool for elucidating the role of GPCR-mediated Ca²⁺ signaling in chondrogenesis and chondrocyte differentiation. Impact statement Targeted activation of intracellular calcium signaling has gained attention as a cartilage tissue engineering adjuvant approach. In the present study, we demonstrated that activation of hM3Dq, an engineered chemogenetic activator of the Gα_q-pathway and IP₃-mediated intracellular calcium signaling, drives accelerated development of mesenchyme-like cell condensations and cartilaginous neotissue formation in chondrocyte-like cell cultures in vitro and does so without the requirement of differentiation factors/inducers. These outcomes highlight the potential of targeted/synthetic Gα_q-pathway activation, specifically using novel chemogenetic approaches, to enhance the study of chondrocyte physiology and improve cartilage tissue engineering approaches.