Mechanical loading and TGF-β change the expression of multiple miRNAs in tendon fibroblasts

Abstract

Tendons link skeletal muscles to bones and are important components of the musculoskeletal system. There has been much interest in the role of microRNA (miRNA) in the regulation of musculoskeletal tissues to mechanical loading, inactivity, and disease, but it was unknown whether miRNA is involved in the adaptation of tendons to mechanical loading. We hypothesized that mechanical loading and transforming growth factor-β (TGF-β) treatment would regulate the expression of several miRNA molecules with known roles in cell proliferation and extracellular matrix synthesis. To test our hypothesis, we subjected untrained adult rats to a single session of mechanical loading and measured the expression of several miRNA transcripts in Achilles tendons. Additionally, as TGF-β is known to be an important regulator of tendon growth and adaptation, we treated primary tendon fibroblasts with TGF-β and measured miRNA expression. Both mechanical loading and TGF-β treatment modulated the expression of several miRNAs that regulate cell proliferation and extracellular matrix synthesis. We also identified mechanosensitive miRNAs that may bind to the 3'-untranslated region of the basic helix-loop-helix transcription factor scleraxis, which is a master regulator of limb tendon development. The results from this study provide novel insight into the mechanobiology of tendons and indicate that miRNA could play an important role in the adaptation of tendons to growth stimuli.

Fig. 1.

RNA isolated from cultured tendon fibroblasts and whole achilles tendons did not contain contaminating RNA from skeletal muscle tissue, as indicated by the absence of expression of the myogenic basic helix-loop-helix transcription factor MyoD.

Fig. 2.

Mechanical loading induces the expression of type I collagen (Col1a1), proliferating cell nuclear antigen (PCNA), scleraxis (Scx), and tenomodulin (Tnmd) in Achilles tendons. Target gene expression is normalized to the stable housekeeping gene GAPDH. Values are means ± SD; n = 5 rats from each group. *P < 0.05, significantly different from sedentary rat Achilles tendons.

Fig. 3.

Mechanical loading changes the expression of several microRNA (miRNA) transcripts associated with cell proliferation and extracellular matrix (ECM) synthesis (A), skeletal muscle adaptation (B), chondrogenesis and neovascularization (C), the let-7 cluster (D), and miRNAs (E) predicted to bind to the 3′-untranslated region (UTR) of scleraxis or tenomodulin. Target miRNA expression is normalized to the stable housekeeping noncoding RNA Rnu6. Values are means ± SD; n = 5 rats from each group. *P < 0.05, significantly different from sedentary rat Achilles tendons.

Fig. 4.

Transforming growth factor-β (TGF-β) treatment induces the expression of type I collagen, PCNA, scleraxis, and tenomodulin in cultured tendon fibroblast cells. Target gene expression is normalized to the stable housekeeping gene GAPDH. Values are means ± SD; n = 5 replicates from each group. *P < 0.05, significantly different from fibroblasts that were not treated with TGF-β.

Fig. 5.

TGF-β treatment changes the expression of several miRNA transcripts associated with cell proliferation and ECM synthesis (A), skeletal muscle adaptation (B), chondrogenesis and neovascularization (C), the let-7 cluster (D), and miRNAs (E) predicted to bind to the 3′-UTR of scleraxis or tenomodulin. Target miRNA expression is normalized to the stable housekeeping noncoding RNA Rnu6. Values are means ± SD; n = 5 replicates from each group. *P < 0.05, significantly different from fibroblasts that were not treated with TGF-β.