Sex-specific regulation of growth plate chondrocytes by estrogen is via multiple MAP kinase signaling pathways.
Both male and female rat growth plate cartilage cells possess estrogen receptors (ERs), but 17beta-estradiol (E(2)) activates protein kinase C (PKC) and PKC-dependent biological responses to E(2) only in cells from female animals. PKC signaling can elicit genomic responses via mitogen activated protein kinase (MAPK) and E(2) has been shown to activate ERK MAPK in many cells, suggesting that MAPK may play a role in growth plate chondrocytes as well. We tested if E(2) increases MAPK activity and if so, whether the response is limited to female cells, if it is PKC-dependent, and if the mechanism involves traditional ER pathways. We also determined the contribution of MAPK to the biological response of growth plate chondrocytes and assessed the relative contributions of ERK, p38 and JNK MAPKs. Female rat costochondral cartilage cells were treated with E(2) and MAPK-specific activity determined in cell layer lysates. The mechanism of MAPK activation was determined by treating the cells with E(2) conjugated to bovine serum albumin (E(2)-BSA) to assess if membrane receptors were involved; stereospecificity was determined using 17alpha-estradiol; PKC and phospholipase C (PLC) dependence was determined using specific inhibitors; and the ER agonist diethylstilbestrol, the ER antagonist ICI 182780, and tamoxifen were used to assess the role of traditional ER pathways. E(2) regulation of ERK1/2 MAPK was assessed and the relative roles of ERK1/2, p38 and JNK MAPKs determined using specific inhibitors. E(2) caused a rapid dose-dependent activation of MAPK that was greatest in cells treated for 9 min with 10(-9) M hormone; activity remained elevated for 3 h. E(2)'s effect on MAPK was stereospecific and comparable to that of E(2)-BSA. It was insensitive to DES and ICI 182780, dependent on PKC and PLC, blocked by tamoxifen and it did not require gene transcription or translation. E(2) had no effect on ERK1 or ERK2 mRNA or protein but it caused a rapid phosphorylation of ERK1/2 at 9 min. Inhibition of ERK1/2 and p38 MAPK reduced the stimulatory effects of E(2) on alkaline phosphatase activity and [(35)S]-sulfate incorporation. These results suggest that E(2) regulates MAPK through a sex-specific membrane-mediated mechanism that does not involve cytosolic ERs in a traditional sense and that ERK1/2 and p38 mediate the downstream biological effects of the hormone.
McMillan
J
J
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 315 Ferst Drive NW, Atlanta, 30332, USA.
Fatehi-Sedeh
S
S
Sylvia
V L
VL
Bingham
V
V
Zhong
M
M
Boyan
B D
BD
Schwartz
Z
Z
D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004958:Estradiol / Q000502:physiology*; D005260:Female; D006132:Growth Plate / Q000166:cytology / Q000201:enzymology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012727:Sex Characteristics
D004958:Estradiol