Estrogen modulation of chondrocyte mechanotransduction via PIEZO1 and TRPV4

Ut Southwestern Medical Center, Dallas TX

Investigators

Abstract

PROJECT SUMMARY For reasons still not well understood, women have a higher prevalence of knee articular cartilage degeneration than men specially after menopause. Sex-specific gait mechanics, muscle strength, metabolism, and sex hormones are key contributors to this health disparity. However, despite growing efforts to reveal sex differences in cartilage, the influence of both the biological sex and sex hormones in modulating cartilage anabolism and catabolism are still unclear, especially during age-related hormonal changes (contraceptive use, menopause, and hormonal replacement therapy in women). As cartilage structure results from chondrocyte mechanotransduction, we propose that the key to sexual dimorphism in cartilage and the exacerbation of risks for catabolism after menopause in women are related to how chondrocytes respond to mechanical stimulation depending on both the biological sex and sex hormones. Defining how biological sex and sex hormones modulate chondrocyte mechanotransduction will help identify new targets to treat and prevent age-related cartilage degeneration, especially in women undergoing hormonal changes. Moreover, answering these questions will be foundational in understanding how sex hormones affect cartilage in gender minorities, specifically the transgender population going through gender-affirming intervention, for whom changes in musculoskeletal health after transition are understudied. The calcium-permeant mechanosensitive channels PIEZO1 and TRPV4 are well-known mediators of chondrocyte mechanotransduction and involved in cartilage anabolism and catabolism. According to our supporting data, their gene expression in response to physiological levels of cyclic compression are influenced by the biological sex and 17-β Estradiol (E2), and E2 decreases the calcium influx induced by the pharmacological activation of TRPV4 and PIEZO1. Therefore, we hypothesize that E2 affects chondrocyte mechanotransduction by regulating the activity and downstream effect of PIEZO1 and TRPV4 in a sex-specific manner. Since we previously found sex differences in the extracellular matrix (ECM) of adult bovine cartilage (equivalent to human premenopausal ages), we will investigate sex differences in chondrocyte mechanotransduction without the confounding effect of the ECM and at premenopausal ages. Here, we will isolate chondrocytes from healthy human cartilage from postmortem donors (20-40 years old) to grow them in 2% agarose discs and test our hypothesis by defining the sex-specific and E2-regulated role on the activation and downstream effect of PIEZO1 and TRPV4 (Aim 1) and defining the effect of biological sex and E2 in chondrocyte mechanotransduction mediated by PIEZO1 and TRPV4 (Aim 2). In Aim 1, we will (1.1) measure calcium intake transients in absence or presence of a premenopausal concentration of E2, and (1.2) perform phosphoproteomic and bulk-RNAseq analysis of chondrocytes treated with specific PIEZO1 and TRPV4 agonists (Yoda-1 and GSK101, respectively) in the absence or presence of E2. In Aim 2, we will stimulate chondrocytes with cyclic compression at physiological levels (8-10% strain) to activate TRPV4 (2.1) or a hyper physiological level (70% strain) to activate PIEZO1 (2.2) in absence or presence of E2. The most significant differentially expressed genes modulated by E2 after activation of each channel in Aim1 will be transiently silenced with siRNA before applying mechanical stimulation. We will assess the expression of selected anabolic and catabolic markers. We propose that testing our hypothesis first in premenopausal aged samples is key to later expand to postmenopausal samples. Data obtained in this E2-focused study will be foundational for a future multi-hormonal study.