Destabilization of F-actin by Mechanical Stress Deprivation or Tpm3.1 Inhibition Promotes a Pathological Phenotype in Tendon Cells

The actin cytoskeleton is a central mediator between mechanical force and cellular phenotype. In tendon, it is speculated that mechanical stress deprivation regulates gene expression by filamentous (F-) actin destabilization. However, the molecular mechanisms that stabilize tenocyte F-actin networks remain unclear. Tropomyosins (Tpms) are master regulators of F-actin networks. There are over 40 mammalian Tpm isoforms, with each isoform having the unique capability to stabilize F-actin sub-populations. Thus, the specific Tpm(s) expressed by a cell defines overall F-actin organization. Here, we investigated F-actin destabilization by stress deprivation of tendon and tested the hypothesis that stress fiber-associated Tpm(s) stabilize tenocyte F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon fascicles downregulated tenocyte genes (collagen-I, tenascin-C, scleraxis, -smooth muscle actin) and upregulated matrix metalloproteinase-3. Concomitant with mRNA modulation were increases in DNAse-I/Phallodin (G/F-actin) staining, confirming F-actin destabilization by tendon stress deprivation. To investigate the molecular regulation of F-actin stabilization, we first identified the Tpms expressed by mouse tendons. Tendon cells from different origins (tail, Achilles, plantaris) express three isoforms in common: Tpm1.6, 3.1, and 4.2. We examined the function of Tpm3.1 since we previously determined that it stabilizes F-actin stress fibers in lens epithelial cells. Tpm3.1 associated with F-actin stress fibers in native and primary tendon cells. Inhibition of Tpm3.1 depolymerized F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin stability in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.