[Stanozolol activates the cross-talk of estrogen receptor alpha-insulin-like growth factor-1 receptor-extracellular-signal regulated kinase 1/2 in the growth plate chondrocytes of estrogen-inhibited adolescent rats in vitro].
OBJECTIVE
To investigate the effects and the mechanisms of stanozolol (ST) on the proliferation, maturation and differentiation of in vitro cultured growth plate chondrocyte isolated from gonadotropin releasing hormone analogue (GnRHa)-treated adolescent rats, to study if ST mediates the proliferation of chondrocytes via the estrogen receptor alpha (ERalpha), androgen receptor (AR) and/or insulin-like growth factor-1 receptor (IGF-1R) and interactions of the two receptor and IGF-1R receptor signaling pathway, to investigate the mechanism of the biological effects in ST promoting bone growth/maturity at molecular level.


METHODS
The rats were weaned at the end of 3 weeks and intramuscular injection of triptorelin of GnRHa preparations, qow x 2 was started. The rats were sacrificed at the end of 7 weeks, and then the tibiae growth plates were taken out with sterile procedure. The chondrocytes were obtained by two-time enzyme digestion method, and the experiments were carried out with the primary chondrocytes. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and Western blot analysis were applied.


RESULTS
The results of PCNA demonstrated that stanozolol enhanced the proliferation of the chondrocytes, time-course studies showed that the proliferation were maximally stimulated by stanozolol after 2 days of incubation and decreased again after longer periods of incubation. The expression of p-ERalpha, p-IGF-1R and p-extracellular-signal regulated kinase 1/2 (ERK1/2) increased with the incubation period of ST treatment, and reached the peak value at a certain time, and then gradually decreased. The expression of p-ERalpha, p-IGF-1R and p-ERK1/2 increased with the elevation of ST concentration, and reached the peak value at 10(-9) - 10(-8) mol/L, then gradually decreased. ST induced-p-ERalpha expression was partially blocked by ERalpha and mitogen-activated protein kinase kinase inhibitors. ST induced-p-IGF-1R expression was partially blocked by ERalpha and IGF-1R inhibitors. ST induced-p-ERK1/2 expression was partially blocked by mitogen-activated protein kinase kinase and IGF-1R inhibitors.


CONCLUSIONS
As an androgen derivation, ST exerts its biological effects of promoting proliferation of the long bone growth plate chondrocytes via activating the classic ERalpha receptor pathway and mitogen-activated protein kinase pathway, and at the same time, by activation of IGF-1R. Both IGF-1R and ERalpha can promote "cross-talk" of two systems' receptor signal through mitogen-activated protein kinase signal pathway.
Zhu
Shun-ye
SY
Department of Pediatrics, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510089, China.
Li
Yan-hong
YH
Ma
Hua-mei
HM
Pan
Si-nian
SN
Chen
Hong-shan
HS
DU
Min-lian
ML
D000728:Androgens / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D006132:Growth Plate / Q000187:drug effects* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D051381:Rats; D020239:Receptor Cross-Talk; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013197:Stanozolol / Q000494:pharmacology*
D000728:Androgens; D047628:Estrogen Receptor alpha; D013197:Stanozolol; D017526:Receptor, IGF Type 1; D048052:Mitogen-Activated Protein Kinase 3