GRK2, a novel regulator of skeletal development
Authors: Yoshioka Natalie K. , Karuppagounder Vengadeshprabhu , Pinamont William J. , Deiter Gina M. , Elbarbary Reyad A. , Pacifici Maurizio , Kamal Fadia 
Published: May 2022

STUDY OBJECTIVE

During skeletal development, the cartilaginous growth plate (GP) allows for bone elongation, composed of chondrocytes organized into three histological zones: resting (RZ), proliferative (PZ), and hypertrophic (HZ). Resting chondrocytes proliferate, become prehypertrophic, then hypertrophic, to create room for bone deposition. The G‐protein coupled receptor (GPCR) Parathyroid Hormone Receptor‐1 (PTH1R) expressed on the chondrocytes of the prehypertrophic zone, decelerates the transition to hypertrophy. PTH1R signaling is regulated by GPCR kinase 2 (GRK2); GRK2 phosphorylates PTH1R leading to its internalization and signal termination. Recent studies showed that loss of function mutations in GRK2 result in skeletal deformities, and we demonstrated that GRK2 promotes chondrocyte hypertrophy (CH) in adult arthritic cartilage. However, the role of GRK2 in molecular signaling in GP chondrocytes has yet to be explored.

HYPOTHESIS

We hypothesized that GRK2 desensitizes PTH1R to promote CH in the GP.

METHODS

We utilized the Cre‐Lox system to generate transgenic mice with inducible ‐conditional‐knockout of GRK2 (GRK2‐icKO) in chondrocytes. KO was initiated at postnatal day 3 (PN3) by Tamoxifen ip injections and mice were sacrificed at PN10 to examine the effects on early and late stages of skeletal development. We performed H&E staining to analyze histological changes in GP structure. To determine changes in molecular signaling, we used ISH and IF staining to quantify expression of GRK2, PTH1R, BrdU (proliferation marker), and ColX (CH marker). At the chondro‐osseous junction, we quantified apoptosis (TUNEL staining), bone deposition (Col1 and Runx2 IF), and matrix mineralization (Alizarin Red (AR) staining). We used in vitro experiments with primary chondrocyte culture to confirm in vivoresults. GRK2 knockdown (KD) was achieved with siRNA before inducing CH. Gene and protein expression of the same molecular markers were analyzed with qPCR and WB, and matrix mineralization quantified by AR.

RESULTS

GKR2‐icKO mice had: (1) Shorter bones (2) Retained PTH1R expression in the HZ, with reduced gene expression (Fig. 1A), as confirmed in vitro (Fig. 1D) (3) Longer PZ and more proliferative chondrocytes confirmed by BrdU, (4) shorter HZ and less hypertrophic chondrocytes, confirmed by ColX IF (Fig. 1B). (5) At the chondro‐osseous junction, GRK2‐icKO mice had reduced chondrocyte apoptosis, bone deposition, and matrix mineralization (Fig. 1C). In vitroexperiments demonstrated increased GRK2 expression with CH, where GRK2 KD reduced CH and matrix mineralization (Fig. 1D).

CONCLUSIONS

We show a novel role of GRK2 in skeletal development as a major regulator of PTH1R in GP chondrocytes. GRK2‐icKO increases chondrocyte proliferation and reduces hypertrophy and apoptosis, leading to reduced bone deposition, matrix mineralization, and shorter bones. Altogether, this presents GRK2 as a novel therapeutic target for skeletal abnormalities.