Steroids 70 (2005) 775–786 Growth-plate chondrocytes respond to 17␤-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism J. Ekstein a , E. Nasatzky a , B.D. Boyan b,∗ , A. Ornoy a , Z. Schwartz a,b,c a Department of Periodontics, Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel b Wallace H. Coulter School of Biomedical Engineering, Georgia Institute of Technology, 315 Ferst Drive NW, Atlanta, GA 30332, USA c Department of Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Received 14 January 2005; received in revised form 26 April 2005; accepted 29 April 2005 Available online 6 July 2005 Abstract 17␤-Estradiol (E 2 ) regulates growth-plate chondrocyte differentiation in a gender and cell maturation-dependent manner via classic nuclear receptors ER␣ and ER␤, 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 17␣-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. © 2005 Elsevier Inc. All rights reserved. Keywords: 17␤-Estradiol; Sex specificity; Chondrocytes; Calcium; Thapsigargin; Verapamil; EGTA 1. Introduction Classically,estrogeneffectsaremediatedthrough genomic mechanisms that involve the diffusion of estro- gen across the plasma membrane and activation of specific intracellular receptors. Binding of 17␤-estradiol (E 2 ) to estro- gen receptor alpha (ER␣) or ER␤ in the cytoplasm results in translocation of the hormone–receptor complex to the nucleus where it interacts with DNA to regulate gene tran- scription [1]. Estrogen is an important regulator of cartilage ∗ Corresponding author. Tel.: +1 404 385 4108; fax: +1 404 894 2291. E-mail address: Barbara.Boyan@bme.gatech.edu (B.D. Boyan). biology. Specific receptors for estrogen have been found in articular cartilage [2,3], growth plate [4,5] and fracture callus [6],and recent studies have shown that growth-plate chon- drocytes also produce estrogen locally via aromatization of androgen [7,8]. E 2 also acts on cells via G-protein-dependent mechanisms [9,10],as wellas through tyrosine kinase signalling path- ways [11]. It has been shown to rapidly stimulate the entry of Ca 2+ into isolated duodenal enterocytes by a phospholipase C (PLC)-dependent mechanism involving store-operated Ca 2+ channels [12]. E 2 -stimulated intracellular Ca 2+ flux via L- type voltage gated Ca 2+ channels has been postulated to be responsible for E 2 -dependent increases in nitric oxide 0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2005.04.007