G-Protein Coupled Receptor Kinase 2: A Novel Regulator of Postnatal Skeletal Development
Restricted (Penn State Only)
- Author:
- Yoshioka, Natalie
- Graduate Program:
- Anatomy (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 06, 2022
- Committee Members:
- Fadia Kamal, Chair & Dissertation Advisor
Patricia Mclaughlin, Major Field Member
James Broach, Major Field Member
Amanda Nelson, Outside Unit & Field Member
Irina Elcheva, Major Field Member
Patricia Mclaughlin, Program Head/Chair - Keywords:
- skeletal development
endochondral ossification
PTHrP-IHH feedback loop
GRK2 - Abstract:
- ABSTRACT STUDY OBJECTIVE: During endochondral ossification, the process by which the majority of bones are formed, a cartilaginous precursor gradually ossifies, initially forming a primary ossification center (POC) at the diaphysis prenatally, and secondary ossification centers (SOC) at the epiphyses postnatally. At the metaphysis, a cartilaginous remnant called the growth plate (GP) allows for continued bone elongation and is composed of chondrocytes organized into several distinct histological zones: resting (RZ), proliferative (PZ), pre-hypertrophic (pre-HZ) and hypertrophic (HZ). Resting chondrocytes enter a proliferative state, then become prehypertrophic and hypertrophic, to create room for bone deposition. This process is regulated by the Parathyroid hormone related protein (PTHrP) / Indian Hedgehog (IHH) feedback loop, in which PTHrP signaling maintains chondrocyte proliferation and IHH accelerates chondrocyte hypertrophy (CH). Parathyroid Hormone Receptor-1 (PTH1R) the receptor for PTHrP, is expressed on the chondrocytes of the prehypertrophic zone to decelerate CH and is essential for GP maintenance. PTH1R is a G-protein coupled receptor (GPCR); signaling is regulated by GPCR kinase 2 (GRK2) which phosphorylates PTH1R, leading to internalization and signal termination. Currently, full mechanisms of GKR2 molecular signaling in GP chondrocytes are unclear. However, our research has shown that inhibition of GRK2 prevents chondrocyte hypertrophy (CH) in adult arthritic cartilage and recent studies demonstrated that loss of function mutations in GRK2 result in skeletal deformities. HYPOTHESIS: Therefore, we hypothesized that GRK2 desensitizes PTH1R to promote CH in the GP. METHODS: We used the Cre-Lox system to perform inducible, conditional knockout of GRK2 (GRK2-icKO) in GP chondrocytes at postnatal day 3 (P3) with Tamoxifen intraperitoneal (ip) injections, sacrificing pups at P10 or P28 to examine the effects on early and late stages of postnatal skeletal development between cre negative (CTRL) and cre positive (GRK2-icKO) littermates. At these same timepoints, we used fate-tracing with Ai9 Cre reporter mice, to tag chondrocytes with flourescent tdTomato and examine their maturation through the GP, comparing Ai9-Ch mice (tagged non-KO CTRL) to Ai9-Ch;GRK2-icKO mice (tagged GRK2-icKO). We evaluated morphology with whole mount skeletal staining and analyzed histological changes in chondrocyte phenotype and GP structure with hematoxylin & eosin (H&E) staining, Safranin-O/ Fast Green staining, and histomorphometry. To examine changes in molecular signaling within the GP, we used immunofluorescence (IF) and in situ hybridization (ISH) staining to quantify expression of GRK2, BrdU (proliferation marker), ColX (CH marker), and PTHrP/IHH feedback loop intermediates. To examine changes in the cartilage – to – bone transition, we quantified apoptosis (TUNEL staining), bone deposition (Runx2 and Col1IF staining), matrix resorption (MMP13), and matrix mineralization (Alizarin Red (AR) staining) at the chondro-osseous junctions and primary spongiosa of the POC and SOC. We used in vitro experiments with primary chondrocyte culture to confirm in vivo results, in which GRK2 knockdown was performed with siRNA transfection prior to inducing CH with differentiation media. Gene and protein expression of the same molecular markers were analyzed with reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB), and chondrocyte phenotype was quantified by AR and Alcian blue (AB) staining. Finally, we explored expression of GRK2, bone markers, and molecular regulators in the secondary ossification center in which CH plays a key role; and in the perichondrium, the connective tissue layer surrounding GP cartilage, which directs formation periosteum and cortical bone postnatally in response to CH. RESULTS: We report a novel finding of GRK2 expression in GP chondrocytes, which peaked in the prehypertrophic zone, and was significantly reduced with GRK2-icKO at both P10 and P28. GKR2-icKO mice at both P10 and P28 had: (1) Shorter bones and skeletal length (2) longer PZ and more proliferative chondrocytes confirmed by BrdU, (3) shorter HZ and less hypertrophic chondrocytes, confirmed by ColX IF. Fate tracing confirmed this delay in maturation of GP chondrocytes at both P10 and P28, as tagged GRK2-icKO chondrocytes were reduced in the pre-HZ to HZ, primary spongiosa, and SOC in comparison to their tagged non-KO CTRLs. At the chondro-osseous junction and primary spongiosa, GRK2-icKO mice had reduced chondrocyte apoptosis, matrix mineralization, and bone deposition, with persistence of cartilaginous matrix due to increased production by GP chondrocytes as matrix resorption was not changed. Mechanistically, GRK2 deletion resulted in retained PTH1R expression in the HZ and SOC, with reduced gene expression and disarranged expression of IHH in the pre-HZ. GRK2 expression was also reduced in the secondary ossification center (SOC) (though not absent), resulting in delayed SOC formation and persistence of chondrocytes and cartilaginous matrix, in combination with reduction in markers of bone deposition, chondrocyte apoptosis, and matrix mineralization. In vitro experiments confirmed results, where GRK2 KD reduced CH, matrix mineralization, and PTHrP/IHH intermediates. In the perichondrium, GRK2 expression was reduced with reduced bone marker expression. CONCLUSIONS: We show a novel role of GRK2 as a regulator of postnatal skeletal development, where it promotes the hypertrophy of GP chondrocytes through PTH1R desensitization to ensure EO. 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.