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

Abstract

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.

Keywords: G-protein coupled receptor activation; cartilage tissue engineering; chemogenetics; chondrocyte calcium signaling; designer receptors exclusively activated by designer drugs.

FIG. 1.

Primary and oscillatory calcium signaling responses of hM3Dq-ATDC5 cells subjected to once-daily 500 nM CNO administration during long-term stimulatory culture. (A) CNO-evoked primary Ca²⁺ responses remained largely unchanged across 14 days of daily stimulatory culture; however, oscillatory Ca²⁺ signaling behaviors changed over time. The number of cells exhibiting 2+ peaks significantly increased through day 7 of culture (compared with days 1 and 14), whereas extensive oscillatory behaviors significantly diminished at day 14; the number of cells exhibiting 5+ peaks remained stable through day 7 (at ∼28–30%), then dropped to ∼11% at day 14; and the number of cells exhibiting 9+ peaks was suppressed from day 3 onward, dropping from ∼14% to <5%. (B) The average number of oscillations per cell gradually diminished through day 7 of culture, dropping from 4.2 peaks/cell to ∼3.5 peaks, then to ∼2.2 peaks/cell at day 14. *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey's post hoc test. CNO, clozapine N-oxide.

FIG. 2.

Temporal dynamics of CNO-induced Ca²⁺ oscillations in hM3Dq-ATDC5 cells during long-term stimulatory culture. (A) Stimulation with 500 nM CNO at day 1 of culture elicited mean primary Ca²⁺ peak intensities of ∼33 a.u., which quickly dropped to ∼15 a.u. at the second peak and decayed linearly to ∼5 a.u. for later peaks. At day 3 of culture and beyond, significantly reduced primary peak intensities (∼13–18 a.u.; p < 0.05, one-way ANOVA with Tukey's post hoc test), which decayed to 2–3 a.u. at later peaks, were observed. (B) Peak-to-peak periods exhibited similar dynamics across all culture timepoints; the first peak interval (i.e., between the primary and initial secondary peak) was always longer than all subsequent intervals, which decayed to a plateau between 25 and 45 s. The initial inter-peak period decreased—non-significantly—from ∼160 s at day 1 to ∼130–135 s at days 3 and 7, and significantly to ∼105 s at day 14 (p < 0.01, one-way ANOVA with Tukey's post hoc test). (C) Mean Ca²⁺ oscillation height decreased significantly from ∼11 a.u. at day 1 to ∼5 a.u. at day 3 and beyond. (D) Mean oscillation peak-to-peak period gradually decreased from ∼116 s at day 1 to ∼108 s at day 3, then to ∼105 s at day 7 and ∼85 s at day 14. *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA with Tukey's post hoc test. The statistical comparisons indicated in red fell just short of significance, p < 0.10.

FIG. 3.

Effect of CNO stimulation on hM3Dq cell proliferation and health during early stimulatory culture. (A) Location-matched DIC images revealed CNO concentration and frequency-dependent increases in the proliferative capacity of hM3Dq cells; elevated stimulatory pressures induced increased cell proliferation compared with less-frequent and lower concentration stimulation; however, all CNO stimulated groups appeared to proliferate faster than the non-CNO groups. (B) Cell viability was quite consistent among all groups after 24 h of culture, at >95% cells. At 72 h, only unstimulated WT cells experienced a—minor—decrease in viability, dropping from 96% to 94%; no significant changes in viability were observed among WT or unstimulated hM3Dq cells; and hM3Dq cells multiply stimulated with CNO or GSK101 saw significant increases in viability at 72 h compared with 24 h, increasing by ∼2–3% percentage points. (C) Apoptosis for all groups increased at 72 h compared with 24 h, but this increase was significantly suppressed in multiply stimulated hM3Dq cells in a concentration- and frequency-dependent manner; the highest CNO-stimulated group (3 × /day 500 nM) displayed <1% apoptosis. (D) Viability and apoptosis in hM3Dq cells stimulated once daily with 500 nM CNO was very high (>92%) and low (<5%), respectively, after 2 weeks of culture. *p < 0.05 compared with WT (& WT+CNO), Kruskal–Wallis test with Dunn's multiple-corrections tests. Bar = p < 0.05 for the comparison between indicated groups for the noted CNO concentration, Kruskal–Wallis test with Dunn's multiple-correction tests. ^#p < 0.05 for comparison between respective 72- and 24-h outcomes, Mann–Whitney test. Statistical comparisons indicated in red are those that fell just short of significance, p = 0.0558 for Kruskal–Wallis test and p = 0.0571 for Mann–Whitney test. DIC, differential interference contrast.

FIG. 4.

CNO stimulation of hM3Dq-ATDC5 cells drove concentration- and frequency-dependent neotissue formation. (A) PR staining demonstrated deep and robust staining for collagens within cell condensations/nodules formed following 2 weeks of CNO stimulatory culture. The size and alignment of tissue nodules increased with CNO concentration and dosing frequency; PR staining of nodules changed from a purple/deep red under lower CNO stimulation regimes to bright red-orange with increased stimulation pressure (reflecting increased collagen content), whereas inter-territorial cells stained light pink/light purple (indicating little to no collagen deposition). Purple/deep red stained cell clumps were observed in GSK101-treated and unstimulated micromass cultures; however, these cells did not exhibit the tissue alignment and condensations observed in the CNO-treated cultures. (B) Similar outcomes were observed for AB staining of proteoglycans; CNO induced concentration- and frequency-dependent enhancements in proteoglycan deposition (blue). GSK101-stimulated cultures developed appreciable AB staining, though this was more diffuse compared with CNO cultures, whereas unstimulated cultures were relatively devoid of AB staining. AB, alcian blue; PR, picrosirius red.

FIG. 5.

Analysis of neotissue nodule geometries and shape descriptors and collagen staining intensity after long-term stimulatory culture (14 days). (A) Average nodule size increased in a CNO dose- and frequency-dependent manner, from ∼20,000 μm² in the least stimulated group to ∼120,000 μm² and ∼320,000 μm² in the thrice-daily 50 and 500 nM CNO-stimulated groups, respectively (p < 0.001). GSK101 and hypo-osmotic shock (HS) areas were indistinguishable from those of WT and unstimulated hM3Dq cultures. Circles having sizes scaled to total nodule areas are provided above the figure for visual reference. (B) The area fraction of nodules increased with CNO concentration and frequency; from ∼4–7% in the 1 × /week groups to ∼10% in the 5 nM CNO group and ∼24–38% in the 50 and 500 nM groups, respectively, when stimulated with CNO thrice daily (p < 0.001). Area fractions for GSK101 and HS-stimulated cultures were indistinguishable from those of WT/unstimulated cultures. (C) Similar CNO dose and frequency-dependent increases in the Feret's diameter of formed nodules (i.e., maximal geometric width) were observed. Circles having diameters and aspect ratios scaled to demonstrate treatment-dependent changes in nodule size and shapes are provided above the figure for visual reference. (D) Analyzing the aspect ratio of formed neotissue nodules demonstrated increasing elongation with increasing CNO dose and stimulation frequency. (E) Changes in collagen staining, that is, PR stain color, brilliance (gray content), and intensity, were assayed via measures of hue, saturation, and value, respectively. With increasing CNO concentration and dose-frequency, the stained collagen-rich tissues exhibited deeper (∼0.7–0.8) and darker (∼0.2–0.4) blue-red (∼350°) colors, indicative of increased collagen deposition. In (E), as well as (A, C), the colors of the indicated circles have been matched to the average HSV values of the group. *p < 0.05 compared with WT (& WT+CNO), bar = p < 0.05 for the comparison between indicated groups for the noted CNO concentration, p < 0.05 compared with once-only activation at the noted CNO concentration, and p < 0.05 compared with the 5 nM CNO activation for the same administration frequency; Kruskal–Wallis test with Dunn's multiple-correction tests. Statistical comparisons indicated in red are those that fell just short of significance, p < 0.1.

FIG. 6.

ICC staining revealed that CNO-mediated activation of hM3Dq enhanced collagen type II and aggrecan deposition. CNO stimulation resulted in the qualitative appearance of concentration-dependent enhancement of antibody staining for collagen type II (top row) and aggrecan (bottom row). Once-daily stimulation with 500 nM CNO exhibited the highest levels of deposition of both proteins, whereas 5 nM CNO displayed increased staining relative to GSK101 and unstimulated cultures; appreciable but reduced staining for collagen type I (middle row) was also detected. The black region in the 1 × /day 5 nM CNO aggrecan image is a “masked-out” fluorescent dust particle.