Journal of Steroid Biochemistry & Molecular Biology 80 (2002) 401–410 Tamoxifen elicits its anti-estrogen effects in growth plate chondrocytes by inhibiting protein kinase C Z. Schwartz a,b,c , V.L. Sylvia a , T. Guinee a , D.D. Dean a , B.D. Boyan a,b,d,∗ a Department of Orthopaedics, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA b Department of Periodontics, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA c Department of Periodontics, Hadassah Faculty of Dental Medicine, Hebrew University, Jerusalem 91120, Israel d Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA Received 3 October 2001; accepted 11 January 2002 Abstract 17-Estradiol (E 2 ) regulates growth plate cartilage cells via classical nuclear receptor mechanisms, as well as by direct effects on the chondrocyte membrane. These direct effects are stereospecific, causing a rapid increase in protein kinase C (PKC) specific activity, are only found in cells from female rats and are mimicked by E 2 -bovine serum albumin (BSA), which cannot penetrate the cell membrane. E 2 and E 2 -BSA stimulate alkaline phosphatase specific activity and proteoglycan sulfation in female rat costochondral cartilage cell cultures, but traditional nuclear receptors do not appear to be involved. This study examined the effect of the anti-estrogen tamoxifen on these markers of chondrocyte differentiation; the gender-specificity of tamoxifen’s effect on PKC, if tamoxifen has an effect on vitamin D metabolite-stimulated PKC, which is mediated via specific membrane receptors (1,25-mVDR; 24,25-mVDR) and whether the effect of tamoxifen is mediated by nuclear estrogen receptors. Tamoxifen dose-dependently inhibited the effect of E 2 -BSA on PKC, alkaline phosphatase and proteoglycan sulfation in confluent cultures of female resting zone (RC) cells and growth zone (GC) (prehypertrophic/upper hypertrophic zones) cells, suggesting that its action is at the membrane and not cell maturation-dependent. Neither the estrogen receptor (ER) antagonist ICI 182780 nor the ER agonist diethylstilbesterol affected E 2 or E 2 -BSA-stimulated PKC in female chondrocytes. Tamoxifen also inhibited the increase in PKC activity due to 1,25-(OH) 2 D 3 or 24R,25-(OH) 2 D 3 in growth plate cells derived from either female or male rats. Inhibition of PKC by tamoxifen may be a general property of membrane receptors involved in rapid responses to hormones. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Chondrocyte cultures; Tamoxifen; 1,25-(OH) 2 D 3 ; 17-Estradiol; Protein kinase C; PKC 1. Introduction There is a growing body of evidence that steroid hor- mones, including 17-estradiol (E 2 ), exert their effects via membrane-mechanisms, in addition to the traditional nuclear receptor pathway [1]. This appears to be the case for growth plate chondrocytes. These cells possess nuclear receptors for E 2 [2], indicating that the hormone elicits a number of its effects through classical estrogen receptor-mediated reg- ulation of gene expression. In addition, some of the effects of E 2 on growth plate chondrocytes are mediated by rapid, membrane-associated mechanisms [3–5]. Using rat costo- chondral chondrocytes as a model for studying agents that modulate cartilage cell differentiation and maturation, we have shown that E 2 causes a direct increase in arachidonic ∗ Corresponding author. Tel.: +1-210-567-6326; fax: +1-210-567-6295. E-mail address: boyanb@uthscsa.edu (B.D. Boyan). acid turnover, phospholipase A 2 activity and membrane fluidity [5]. The membrane-mediated effects of steroid hormones contribute to the physiological responses of the target cells via signaling pathways used by peptide hormones, includ- ing PKC [6–9] and MAP kinase [10]. E 2 stimulates protein kinase C (PKC) activity in growth plate chondrocytes from female, but not male rats [4]. This effect is independent of new gene expression and occurs via a pathway that in- volves G-protein-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) [3]. Similarly, gender-specific activation of PKC has been reported in female human colon cancer cells [11]. PKC appears to be involved in the biolog- ical effects of E 2 as well. In growth plate cells, inhibition of PKC with chelerythrine blocks the effects of E 2 on pro- teoglycan sulfation and differentiation, but has no affect on E 2 -dependent proliferation of these cells. In contrast, PKC inhibitors block the action of E 2 on DNA synthesis in a number of cell types [12,13]. 0960-0760/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0960-0760(02)00038-9