Quantitation of Estrogen Receptors and Relaxin Binding in Human Anterior Cruciate Ligament Fibroblasts

Abstract

The significantly higher incidence of anterior cruciate ligament (ACL) injuries in collegiate women compared with men may result from relative ligament laxity. Differences in estrogen and relaxin activity, similar to that seen in pregnancy, may account for this. We quantified estrogen receptors by flow cytometry and relaxin receptors by radioligand binding assay in human ACL cells and compared the presence of these receptors in males and females. ACL stumps were harvested from seven males and eight females with acute ACL injuries. The tissue was placed in M199 cell culture medium. Outgrowth cultures were obtained, and passage 2 cells were used for all studies. Estrogen receptor determination was performed using flow cytometry. Relaxin binding was performed in ACL cells derived from five female and male patients using I(125)-labeled relaxin. Estrogen receptors were identified by flow cytometry in 4 to 10% of ACL cells. Mean fluorescence of cells expressing estrogen receptors was approximately twice that of controls, with no significant differences between males and females. Relaxin studies showed low-level binding of I(125)-relaxin-labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

Osteoarthritis associated with estrogen deficiency

“…Fibroblasts from human ACL, medial cruciate ligament and patellar tendon express functional ER transcripts. Indeed, 4 to 10% of ACL cells express ERs in patients with acute ACL injuries, approximately twice the proportion found in control subjects [13,71]. In human skeletal muscle, ERα mRNA expression was 180-fold higher than that of ERβ [72].…”

Relaxin induces matrix-metalloproteinases-9 and -13 via RXFP1: Induction of MMP-9 involves the PI3K, ERK, Akt and PKC-ζ pathways

“…Both relaxin receptors and their transcripts have been identified in reproductive and non-reproductive tissues such as the brain, kidney, lung, anterior cruciate ligament of the knee joint and in synovial joint fibrocartilaginous cells (Faryniarz et al, 2006; Hsu et al, 2002; Wang et al, 2009). Although the precise contributions of RXFP1 and RXFP2 to modulation of MMPs upon activation by relaxin are not known, indirect evidence for the role of RXFP1 to in vivo remodeling of matrices is provided by the phenotypic characteristics of the female RXFP1 null mice that are similar to those described for relaxin-deficient mice (Kamat et al, 2004; Krajnc-Franken et al, 2004; Zhao et al, 1999).…”

Relaxin's Involvement in Extracellular Matrix Homeostasis

“…Several histologic studies have shown that relaxin binds with specificity to tissue samples of the anterior cruciate ligament. [7][8][9] Initially considered a potential risk factor for female athletic injuries, currently no evidence has accrued to suggest that high serum levels of relaxin contribute to anterior cruciate ligament injury. 10 While anecdotal observations have noted increased joint laxity associated with pregnancy, 11 it remains unclear whether joint hypermobility, as a singular or cumulative event, causes injury or pathology.…”