DETERMINING THE ROLE OF G-PROTEIN RECEPTOR KINASE 2 ON PARATHYROID HORMONE RECEPTOR SIGNALING IN OSTEOARTHRITIS
Open Access
- Author:
- Pinamont, William
- Graduate Program:
- Anatomy
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 19, 2019
- Committee Members:
- Fadia A Kamal, Thesis Advisor/Co-Advisor
David L Waning, Committee Member
Reyad Elbarbary, Committee Member
Patricia Mclaughlin, Committee Member - Keywords:
- Osteoarthritis
Parathyroid Hormone
G protein receptor kinase
Parathyroid Hormone Receptor - Abstract:
- Osteoarthritis (OA) is a complicated and multivariable degenerative disease of diarthrodial joints characterized by the principle cellular process of chondrocyte hypertrophy leading to cartilage degeneration. Although there is no curative treatment to prevent cartilage degeneration in OA, parathyroid hormone (PTH) has previously demonstrated chondroprotective and chondroregenerative effects in mice. PTH binds and signals via parathyroid hormone receptor type-1 (PTH1R), a G-protein coupled receptor (GPCR) expressed in growth plate and articular chondrocytes. In growth plate chondrocytes, PTH-PTH1R-Gs signaling maintains chondrocyte homeostasis and prevents their hypertrophy. G-protein receptor kinases (GRKs) are intracellular proteins that facilitate receptor phosphorylation for internalization and recycling. GRK2 is a GRK that is ubiquitously expressed throughout various cell types, including articular chondrocytes. Elevated GPCR-GRK2 signaling has been described as a contributing factor toward pathological development in other diseases due to GPCR desensitization and the loss of coupled G homeostatic signaling. Inhibition of GRK2 with paroxetine attenuates disease progression in heart and kidney disease however, the role of GPCR-GRK2 signaling in OA remains unknown. We hypothesized that elevated GRK2 signaling in articular chondrocytes leads to PTH1R desensitization and subsequent loss of PTH chondroprotective signaling in OA. Therefore, we predicted GRK2 inhibition by paroxetine will rescue PTH1R chondroprotective signaling and attenuate chondrocyte hypertrophy in OA. This study aims to determine the role of GRK2 inhibition on PTH1R resensitization and PTH chondroprotection in OA through preservation or enhancement of chondrogenic PTH-PTH1R-Gs signaling. Destabilization of the medial meniscus (DMM) surgery was used to simulate clinically relevant post-traumatic OA (PTOA) in our in vivo mouse model. First, we determined the role of GRK2 inhibition in recovering OA cartilage following treatment with high or low dose paroxetine. Then, we investigated the role of GRK2 inhibition in recovering or potentiating PTH1R signaling in OA cartilage by administering PTH and paroxetine alone or together in combination. Finally, we evaluated the effect of a GRK2 inducible conditional knockout (cKO) in articular chondrocytes on cartilage chondroprotection and PTH-PTH1R signaling. Additionally, in vitro experiments using a pre-chondrogenic ATDC5 cell line were performed to evaluate changes to matrix mineralization as well as hypertrophic and chondrogenic gene expression following treatment with PTH and paroxetine. GRK2 inhibition with paroxetine preserved articular cartilage area and prevented chondrocyte hypertrophy alone and also demonstrated an enhanced anabolic effect of paroxetine and PTH in combination. The additive therapeutic effect demonstrated in nonknockout mice treated with PTH and paroxetine in combination suggests a potentiation of PTH-PTH1R chondroprotective signaling as a result of GRK2 inhibition by paroxetine. GRK2 cKO mice demonstrated preservation of cartilage area and attenuation of chondrocyte hypertrophy, however PTH chondroprotective signaling was lost in GRK2 conditional knockout mice, demonstrating a potential threshold of required GRK2 signaling to coordinate to receptor sensitization. Ex vivo analysis of mouse articular chondrocytes demonstrated PTH1R in articular chondrocytes was desensitized in DMM OA mice and PTH1R was resensitized through GRK2 inhibition by paroxetine. Thus, an intracellular balancing of GRK2 signaling seems to play a crucial role in regulating PTH1R sensitization and chondrocyte hypertrophy in OA. Substantial inhibition of GRK2 may disrupt normal receptor recycling and turnover, crucial to maintaining ligand binding responses. In conclusion, GRK2 signaling demonstrated a critical role in PTH chondroprotective signaling in OA. GRK2 inhibition recovers PTH1R signaling and potentiates PTH chondroprotection in OA, demonstrating the potential for a promising therapeutic development. Further understanding of chondrocyte signaling regulation will aid in the development of therapeutic options for osteoarthritis patients. Current treatments for OA primarily focus on palliative pain management therapies with little potential to prevent further articular cartilage degeneration. PTH is currently clinically prescribed for osteoporosis or patients with low bone density and high risk of fracture, however a therapeutic repurposing of the drug has identified a therapeutic effect in OA. Our laboratory team has identified a role of GRK2 signaling in PTH-PTH1R chondroprotective signaling in a PTOA murine model of OA. Future investigations will elucidate the molecular mechanism and downstream signaling changes contributing toward the preservation of chondrocyte homeostasis and prevention of OA progression. Ultimately our goal is to establish a therapeutic advancement in cartilage preservation to reduce patient comorbidities, minimize palliative opioid usage as pain management therapy, and improve overall patient mobility and quality of life for individuals dealing with osteoarthritis.