Matrix stiffness regulates Mkx expression in rat tenocyte through TRPM7

Abstract

Tendon is the fibrous tissue that connects skeletal muscle and bone, playing a crucial role in transmitting forces generated in muscles to bones and thereby facilitating locomotion. Tendon is continuously subjected to mechanical stimuli, such as tensile force and shear stress, and it is well documented that tendon cells respond to these forces and modulate gene expression and tissue structures. However, whether or how tenocytes respond to matrix stiffness, another key mechanical cue for the tissue, remained elusive. While previous studies have shown that mesenchymal stem cells (MSCs) or tendon derived stem cells (TDSCs) modulate tenogenic gene expression in response to stiffness, its effect on tendon fibroblasts was unclear. In this study, we investigated the role of matrix stiffness on tenocytes derived from tail and Achilles tendon of young rats. Tenocytes displayed stiffness-dependent difference in expression of key tendon-related genes, including Mkx, particularly at 40 kPa stiffness. Interestingly, the transient receptor potential melastatin 7 (TRPM7) channel was identified as an upstream regulator of stiffness-dependent Mkx expression. TRPM7 expression was elevated at 40 kPa stiffness, and its knockdown reduced Mkx expression while abolishing the stiffness-dependent expression pattern. This regulation likely occurs through intracellular calcium (Ca²⁺) and/or magnesium (Mg²⁺) ion influx, as Mkx expression was promoted upon Ca²⁺ ionophore treatment or elevation of extracellular Mg²⁺ concentration. This study underscores the importance of stiffness in tendon biology and adds a novel layer to the transcriptional regulation of Mkx, with implications for understanding tendon development, maintenance, and mechanotransduction.