Growth-plate chondrocytes respond to 17β-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism

Abstract

17β-Estradiol (E 2) regulates growth-plate chondrocyte differentiation in a gender and cell maturation-dependent manner via classic nuclear receptors ERα and ERβ, and membrane-associated signalling. Here we show that sex-specific effects of E 2 involve changes in intracellular calcium concentration (ICCC). Resting-zone chondrocytes (RC) and growth-zone chondrocytes (GC) were isolated from costochondral cartilage of male and female rats. Confluent cultures were treated with 10 −8 M E 2 or 17α-estradiol in the presence of high and low extracellular Ca 2+ concentration. The ICCC was determined using laser scanning confocal microscopy to measure changes in Fluo-4 fluorescence every 5 s for a total of 500 s. E 2 increased ICCC in the cells from female rats but had no effect on ICCC in male cells. The effect was rapid (peak at 140 s) and stereospecific. E 2 increased ICCC in RC and GC chondrocytes but the effect was greater in RC cells. Low Ca 2+ media did not abolish the E 2-dependent ICCC elevation, nor did inclusion of verapamil, which inhibits Ca 2+ channels on the cell membrane. Thapsigargin reduced the effect of E 2 on ICCC, showing that Ca 2+ pumps on the endoplasmic reticulum were involved. Pre-treatment of the cells with the ER antagonist ICI 182780 did not alter the stimulatory effect of E 2, suggesting that traditional estrogen receptor mechanisms do not play a role. E 2 caused rapid production of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) but only in female cells, and the effect was greater in RC chondrocytes. These results indicate that E 2 regulates ICCC in a sex-specific and cell maturation state-dependent manner. The mechanism is membrane-associated and is mediated by PLC-dependent IP3 production and release of Ca 2+ from the endoplasmic reticulum.