Estrogen and mechanical loading-related regulation of estrogen receptor-β and apoptosis in tendinopathy

Abstract

Female-dominant tendinopathies are musculoskeletal disorders caused by repetitive hand posture and motion; they are considered overuse syndromes. Both external mechanical stress and changes in hormone levels might affect disease progression. We have previously reported that estrogen receptor-β (ER)-β expression was associated with the pathogenesis of de Quervain's disease. To study the underlying mechanisms, a cyclic stretching culture system was applied to tendon tissue from ovariectomized (OVX) rats. Furthermore, a collagenase I-induced rat tendinopathy model was established to examine the association of ER-β with disease progression. Our results showed that ER-β expression and the number of apoptotic cells were higher and associated with disease severity in rats with tendinopathy. Mechanical stress altered the morphology of primary tenocytes and collagen fiber alignment in tendons, and up-regulated the expression of matrix metalloproteinase-9, ER-β, and interleukin-1β, as well as induced apoptosis in tenocytes and tendon tissue from OVX rats. This is the first report on the effects of ER-β and mechanical stress in tendinopathy. We hope these findings contribute to new pharmacological therapies targeting ER-β signaling pathways to treat tendon-related diseases.

Fig 1. Estrogen receptor-β (ER-β) expression and apoptosis in tendon tissue from rats with tendinopathy.

Adult female Sprague-Dawley rats (200~300 g) were treated with ultrasound-guided intra-tendinous injection of collagenase I into their Achilles tendons for 1,4, and 8 weeks. A, Immunohistochemical staining of ER-β and the number of ER-β-positive cells (n = 15). B, TUNEL staining and the number of TUNEL-positive cells (n = 5~8) in tendon tissue from rats with different grades of tendinopathy. Scale bars represent 50 μm in × 400 magnifications. Data are mean ± standard error of the mean (SEM). R represents Spearman correlation coefficients.

Fig 2. ER-β, interleukin (IL)-1β, matrix metalloproteinase (MMP)-9 expression and apoptosis in cyclically stretched rat primary tenocytes.

A, Representative morphological images of static and mechanically strained tissue (left) and cultured primary tenocytes (middle), as determined by hematoxylin and eosin (H&E) staining. Scale bars represent 100 μm in × 200 magnifications. Expression of α-tubulin (right) in tenocytes after cyclic stretching, as determined by immunofluorescence staining. B, TUNEL assay in tenocytes after cyclic stretching. Nuclei were counterstained with DAPI. C, ER-β, IL-1β, and MMP-9 expression in tenocytes after cyclic stretching, as determined by immunofluorescence staining. D, Cleaved caspase 3 expression in anti-ER-β and isotype control antibody (IgG)-treated tenocytes during cyclic stretching for 24h, as determined by immunofluorescence staining.

Fig 3. Expression of ER-β in tendon tissue from ovariectomized rats after cyclic stretching.

An ovariectomy in adult female Sprague-Dawley rats (200~300 g) was performed and their Achilles tendons were isolated and applied to the following stretch protocol: 15%, 2Hz, time period: 24h A, Serum samples were collected every other day and determined the estrogen levels by ELISA. Ovx, rats were performed with ovariectomy. B, Expression of ER-β and C, calculated ER-β-positive cells in tendon tissue from ovariectomized rats with and without cyclic stretching. Data are mean ± SEM (n = 3). **p<0.01, ***p<0.001.

Fig 4. Cleaved caspase 3 expression and apoptosis in tendon tissue from ovariectomized rats with and without cyclic stretching.

A, Immunohistochemical staining of cleaved caspase 3 and calculated cleaved caspase 3-expressed areas. B, TUNEL staining and the number of TUNEL-positive cells in tendon tissue from ovariectomized rats after cyclic stretching. Scale bars represent 100 and 50 μm in × 200 and × 400 magnifications. Values are the mean ± SEM (n = 3). *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Expression of collagen type I (COLI), matrix metalloproteinase (MMP)-9, and IL-1β in tendon tissue from ovariectomized rats after cyclic stretching.

A, Immunohistochemical staining of COLI and calculated COLI-expressed area B, MMP-9 and calculated MMP-9-expressed area and C, IL-1β and calculated IL-1β-expressed area in tendon tissue from ovariectomized rats after cyclic stretching. Scale bars represent 100 μm in ×200 magnifications. Values are SEM (n = 3). *p<0.05, **p<0.01.