Female hormone receptors are differentially expressed in mouse fibrocartilages

Abstract

Objective: Despite the female predilection for joint diseases, and the known effects of female hormones in regulating chondrocyte function, the various female hormone receptor subtypes in joints are not well characterized, and comparisons in receptor profiles between joints and genders are lacking. This investigation characterized and compared the relative levels of estrogen receptors (ER)-alpha and -beta, relaxin receptors LGR7 and LGR8, and progesterone receptor (PR) in the temporomandibular joint (TMJ) disc, knee meniscus (KM) and pubic symphysis fibrocartilages.

Methods: Fibrocartilaginous cells from 12-week-old mice were maintained in serum-containing alpha-modified Eagle's medium (MEM) until confluence. Total RNA and cell lysates were assayed by RT-PCR, qRT-PCR, immunocytochemistry and Western blots, and joint sections subjected to immunohistochemistry.

Results: All hormone receptors assayed were present in the three joints, but showed substantial differences in expression levels between joints. TMJ cells had higher ER-alpha (>2.8-fold), ER-beta (>2.2-fold), LGR7 (>3-fold) and PR (>1.8-fold), and lower LGR8 (0.5-fold) gene expression levels than KM cells. The ratio of ER-alpha:ER-beta and LGR7:LGR8 was 1.8- and 7.5-fold higher, respectively, in TMJ than in KM cells. The profile of hormone receptors in the TMJ disc was similar to those in the pubic symphysis. Immunochemistry confirmed the differential expression patterns of these receptors in the three tissues. The TMJ cells demonstrated sexual dimorphism in the levels of both ER isoforms, but not of LGR7, LGR8 or PR.

Conclusions: The findings suggest that these fibrocartilages are putative target tissues for actions of female hormones. The differential expression profiles of the hormone receptors in the three joint fibrocartilages and the sexual dimorphism in ERs in TMJ disc cells are likely to result in varied downstream effects in response to hormones within these fibrocartilaginous tissues.

Figure 1

Localization of estrogen receptors ER-α and ER-β, relaxin receptors LGR7 and LGR8, and progesterone receptor PR in mouse TMJ disc, knee meniscus (KM), and pubic symphyseal (PS) fibrochondrocytes. ER-α, ER-β, LGR7 and PR were highly expressed in the TMJ and pubic symphysis fibrochondrocytes while LGR8 was detected at higher levels in knee meniscus and pubic symphysis than in TMJ cells. Negative controls (NC) show pubic symphysis cells subjected to this assay in the absence of primary antibody. The nuclei were stained with DAPI. (Magnification 40×; Bar = 50 μm).

Figure 2

In situ immunolocalization of estrogen receptors ER-α and ER-β, relaxin receptors LGR7 and LGR8 and progesterone receptor PR in mouse TMJ, knee meniscus (KM), and pubic symphysis (PS) fibrocartilages. ER-α, ER-β, LGR7 and PR were more highly expressed in the TMJ and pubic symphysis fibrocartilages but only a few positively stained cells were found in the knee meniscus fibrocartilage. In contrast, LGR8 was mainly expressed in the cells of knee meniscus and pubic symphysis. Negative controls (NC) show pubic symphysis tissues subjected to this assay in the absence of primary antibody. Arrows point to positively stained cells. (Magnification 20×; Bar = 50 μm).

Figure 3

Quantitative differential gene expression for estrogen receptors, ER-α (A), ER-β (B), relaxin receptors, LGR7 (C) and LGR8 (D), and progesterone receptor PR (E) in female mouse TMJ disc, knee meniscus (KM) and pubic symphysis (PS) fibrochondrocytes. ER-α, ER-β, LGR7 and PR were highly expressed in the TMJ and pubic symphysis cells, while LGR8 was highly expressed in knee meniscus cells. Data represents means ± SD fold levels of respective receptors relative to those in the knee meniscus from three independent experiments, each performed in triplicate. (n=3; *p < 0.01, ** p< 0.05 for gene expression of estrogen receptors, relaxin receptors, or progesterone receptor in TMJ or pubic symphysis versus knee meniscus cells).

Figure 4

Differential expression of estrogen receptors ER-α and ER-β, relaxin receptors LGR7 and LGR8, and progesterone receptor PR in mouse TMJ, knee meniscus (KM), and pubic symphysis (PS) fibrochondrocytes. (A). Total RNA, standardized for subjected to semi-quantitative RT-PCR for hormone receptors. GAPDH was used as an internal control. (B) Alternatively, cell lysates, standardized for total protein, were subjected to Western blot assays for each of the hormone receptors. Actin was used as an internal control for the Western blots. Both the genes and protein for ER-Mα, ER-β, LGR-7, and PR were highly expressed in the TMJ and pubic symphysis fibrochondrocytes and were weakly expressed in the knee meniscus cells. In contrast, higher levels of LGR8 were detected in knee meniscus and pubic symphysis cells as compared to that in TMJ cells.

Figure 5

TMJ, knee meniscus (KM) and pubic symphysis (PS) fibrochondrocytes show distinctly different ratios of gene levels for estrogen receptors ER-α:ER-β (A), and relaxin receptors LGR7:LGR8 (B). A significantly higher expression ratio of estrogen receptors ER-α:ER-β and relaxin receptors LGR7:LGR8 was found in the TMJ and pubic symphysis fibrochondrocytes than in knee meniscus cells. Data represents means ± SD from three independent experiments, each performed in triplicate. (n=3; *p < 0.01, **p < 0.05 for ER-α:ER-β or LGR7:LGR8 expression ratio in TMJ or pubic symphysis versus knee meniscus cells).

Figure 6

Gene expression levels for estrogen but not relaxin or progresterone receptors demonstrate sexual dimorphism in mouse TMJ fibrochondrocytes. Hormone receptor gene expression levels in male cells were standardized to that in female cells. Male TMJ cells had a significantly lower gene expression levels for ER-α (67.3%) and ER-β (24.9%), but not for LGR7 (88.7%), LGR8 (75.9%) and PR (116%) relative to that in female TMJ cells. Data represents means ± SD from three independent experiments, each performed in triplicate. (n=3; *p< 0.01, **p< 0.05 for gene expression of estrogen receptors, relaxin receptors, or progesterone receptor in male versus female cells).