OBJECTIVETo investigate the effect of estrogen on cell proliferation and expression of proteins of C-type natriuretic peptide (CNP), natriuretic peptides B receptor (NPR-B) and natriuretic peptides C receptor (NPR-C) in ATDC5 cells during chondrogenesis.

METHODATDC5 cells were induced for differentiation with insulin 10 µg/ml (day 0), and were started to be investigated on day 6. They were incubated with: (1) Estradiol (E2) at different concentrations (10(-11)-10(-5) mol/L) for 24 hours (for studying cell proliferation), or for 48 hours (for studying CNP, NPR-B and NPR-C protein expression); (2) E2 (10(-8) mol/L) for 24, 48, 72, 96 and 120 h (for studying cell proliferation), or for 24, 48, 72 and 96 hours (for studying CNP, NPR-B and NPR-C protein expression); (3) E2 (10(-8) mol/L) , and/or ICI 182782 (estrogen receptor antagonist ) (10(-7) mol/L) for 24 hours (for studying cell proliferation). ATDC5 cells proliferation were determined by MTT (OD value). Western-blotting was performed to identify the protein levels of CNP, NPR-B and NPR-C.

RESULT(1) After incubation with E2 (10(-11)-10(-5) mol/L) for 24 h, ATD5 cell number increased with the increasing E2 concentration, peak in E2 concentrations of 10(-9) and 10(-8) mol/L (0.56 ± 0.06 and 0.52 ± 0.02, P < 0.05 and <0.01, respectively) , while significantly decreased in E2 (10(-5) mol/L) (0.30 ± 0.02) compared with DMSO-control (0.38 ± 0.02) (P < 0.05). After incubation with E2 (10(-11)-10(-5) mol/L) for 48 h, the protein level of CNP, NPR-B and NPR-C increased significantly, with the greatest effect seen at a concentration of 10(-10) mol/L E2 for CNP and NPR-B, 10(-9) mol/L E2 for NPR-C (P < 0.05). (2) After incubation with E2 (10(-8) mol/L) for 24 to 96 hours: (1) The cell number in each of the four time points was significantly increased compared with DMSO-control, with the greatest effect in 48 h (0.030 ± 0.003) (P < 0.05 or <0.01, respectively). While the cell number at 120 h was similar to that in DMSO-control. (2) The protein level of CNP increased significantly at 24 h (P < 0.05), seemed to be increased at 48 h and 72 h and decreased at 96 h. Both NPR-B and NPR-C level seemed to be increased at 24 h (P = 0.060 and 0.055, respectively) and seemed to decrease at 48 h, with decreasing significantly at both 72 h and 96 h (P < 0.05). (3) After incubation for 24 h, there was significant difference among the cell number of the four groups (P < 0.05). Cell number of group E2 (0.470 ± 0.032) was increased compared with group (E2+ICI) (0.410 ± 0.018), both being increased compared with group DMSO-control (0.370 ± 0.011, P < 0.05, respectively). There was no difference in cell number between group ICI 182782(0.360 ± 0.035) and group DMSO-control.

CONCLUSIONE2 promotes the proliferation of ATDC5 cells i.e. chondrogenesis via estrogen receptor mediated mechanism, in both concentration-dependent and time-dependent manner. E2 (10(-11)-10(-8) mol/L) up-regulates protein expression of CNP, NPR-B and NPR-C of ATDC5 cells during chondrogenesis, and regulate the expression of the three proteins mentioned above positively or negatively at different time point, which implied that estrogen is one of the regulators of CNP signaling pathway.