Sex-steroid regulation of relaxin receptor isoforms (RXFP1 & RXFP2) expression in the patellar tendon and lateral collateral ligament of female WKY rats

Abstract

The incidence of non-contact knee injury was found higher in female than in male and is related to the phases of the menstrual cycle. This raised the possibility that female sex-steroids are involved in the mechanism underlying this injury via affecting the expression of the receptors for relaxin, a peptide hormone known to modulate ligament laxity. Therefore, this study aims to investigate the effect of sex-steroids on relaxin receptor isoforms (RXFP1 & RXFP2) expression in the ligaments and tendons of the knee.

Methods: Ovariectomized adult female WKY rats were treated with different doses of estrogen (0.2, 2, 20 μg/kg), progesterone (4mg) and testosterone (125 & 250μg/kg) for three consecutive days. At the end of the treatment, the animals were sacrificed and the patellar tendon and lateral collateral ligament were harvested for mRNA and protein expression analyses by Real Time PCR and Western blotting respectively.

Results: RXFP1, the main isoform expressed in these knee structures and RXFP2 showed a dose-dependent increase in expression with estrogen. Progesterone treatment resulted in an increase while testosterone caused a dose-dependent decrease in the mRNA and protein expression of both relaxin receptor isoforms.

Discussion: Progesterone and high dose estrogen up-regulate while testosterone down-regulates RXFP1 and RXFP2 expression in the patellar tendon and lateral collateral ligament of rat's knee.

Conclusion: Relaxin receptor isoforms up-regulation by progesterone and high dose estrogen could provide the basis for the reported increase in knee laxity while down-regulation of these receptor isoforms by testosterone could explain low incidence of non-contact knee injury in male.

Keywords: RXFP1; RXFP2; collateral ligaments.; patellar tendon; sex-steroids.

Figure 1

The expression of RXFP1 protein in the total homogenate of lateral collateral ligament from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone, LCL-lateral collateral ligament. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test, p= 0.319732. A dose-dependent increase in RXFP1 protein expression was observed following estrogen treatment. Progesterone caused the highest increase while testosterone caused a decrease in this isoform expression. The image was cut in order to arrange the bands in sequence with the label on the x-axis of the bar chart.

Figure 2

The expression of RXFP1 mRNA in lateral collateral ligament from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone, LCL-lateral collateral ligament. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test, p= 0.313319. A dose-dependent increase in RXFP1 mRNA expression was noted with increasing doses of estrogen. Progesterone treatment caused the highest increase while testosterone caused a dose-dependent decrease in RXFP1 mRNA expression

Figure 3

The expression of RXFP1 protein in the total homogenate of the patellar tendon from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone PT- patellar tendon. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test, p= 0.282109. A marked increase in the expression of RXFP1 protein occurs following treatment with high doses of estrogen (20 & 50μg/kg). Meanwhile, progesterone treatment caused the highest increase while testosterone caused a decrease in RXFP1 protein expression in the tendon. The image was cut in order to arrange the bands in sequence with the label on the x-axis of the bar chart.

Figure 4

The expression of RXFP1 mRNA in the patellar tendon from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone, PT-patellar tendon. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test, p= 0.207574. A dose-dependent increase in RXFP1 mRNA was noted following increasing dose of estrogen. Meanwhile progesterone treatment resulted in the highest increase while testosterone reduced the mRNA expression.

Figure 5

The expression of RXFP2 protein in the total homogenate of the lateral collateral ligament from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone, LCL-lateral collateral ligament. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test, p= 0.776415. The expression of RXFP2 protein was increased following treatment with estrogen at doses exceeding 2μg/kg. Meanwhile, progesterone caused the highest increase while testosterone caused a decrease in RXFP2 protein expression. The image was cut in order to arrange the bands in sequence with the label on the x-axis of the bar chart.

Figure 6

The expression of RXFP2 mRNA in the lateral collateral ligament from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone, LCL-lateral collateral ligament. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test, p= 0.668854. Treatment with estrogen at doses exceeding 2μg/kg resulted in a dose-dependent increase in RXFP2 mRNA expression. Meanwhile, progesterone caused the highest increase while testosterone treatment resulted in a decrease in this isoform mRNA expression.

Figure 7

The expression of RXFP2 protein in the total homogenate of the patellar tendon from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone, PT-patellar tendon. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test, p= 0.736386. An increase in the expression of RXFP2 protein was noted following treatment with estrogen exceeding 2μg/kg. Progesterone treatment resulted in the highest increase while testosterone caused a dose-dependent decrease in the expression of this protein isoform. The image was cut in order to arrange the bands in sequence with the label on the x-axis of the bar chart.

Figure 8

The expression of RXFP2 mRNA in the patellar tendon from ovariectomised rats treated with different sex-steroids: C- control; 0.2E - 0.2μg/kg estrogen; 2E - 2μg/kg estrogen, 20E - 20μg/kg estrogen; 50E -50μg/kg estrogen; P - 4mg progesterone; 125T - 125μg testosterone; 250T - 250μg testosterone, PT-patellar tendon. Data were expressed as mean ± SEM, n=6 per treatment group. * p<0.05 as compared to control, Shapiro-Wilk test,p= 0.842243. An increase in the RXFP1 mRNA was noted following treatment with estrogen at doses exceeding 2μg/kg. Progesterone treatment resulted in the highest increase while testosterone caused a dose-dependent decrease in RXFP2 mRNA expression.