Regulation of a Novel Immediate Early Response Gene, IEX-1, in Keratinocytes by 1,25-Dihydroxyvitamin D 3 Teruaki Kobayashi,† Mark R. Pittelkow,* , † ,1 Gina M. Warner,* Karen A. Squillace,† and Rajiv Kumar* , ‡ ,1 *Departments of Medicine, †Dermatology, and ‡Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Received September 14, 1998 1,25-Dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] regulates cellular growth and differentiation. We show that in ker- atinocytes, 1,25(OH) 2 D 3 reduces concentrations of the messenger RNA of IEX-1, the product of which blocks Fas- or tumor necrosis factor type -induced apoptosis in various cells. In sub-confluent keratinocyte cultures, the addition of 1,25(OH) 2 D 3 , in amounts that induce growth arrest, reduces IEX-1 mRNA concentrations. In confluent cells, 1,25(OH) 2 D 3 initially reduces and then increases IEX-1 mRNA concentrations. IEX-1 protein is localized in the nucleus and perinuclear region of kera- tinocytes. In sub-confluent cells, 1,25(OH) 2 D 3 translo- cates IEX-1 protein from the nucleus to the perinuclear region and cytoplasm. Since IEX-1 has recently been shown to regulate cell survival and number, we suggest that IEX-1 may play a role in keratinocyte growth and differentiation and that 1,25(OH) 2 D 3 may reduce kera- tinocyte growth via a reduction in IEX-1 mRNA and a change in the intracellular distribution of IEX-1 protein. © 1998 Academic Press 1,25-Dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] plays an important role in cellular growth and differentiation (1-8). When cells such as keratinocytes and breast can- cer cells, grown in the presence of serum, and treated with 1,25(OH) 2 D 3 , a retardation in cellular growth is generally observed (9-12). At the same time, cells de- velop a more differentiated phenotype (10-13). In the absence of serum and in defined culture media, low concentrations of 1,25(OH) 2 D 3 increase cellular growth rate (10,14). The mechanisms by which 1,25(OH) 2 D 3 alters cellular growth are varied and have not completely defined (15-30). Several investiga- tors have shown that growth factors play an important role in this process (9,11,21-33). For example, in kera- tinocytes and osteoblasts, 1,25(OH) 2 D 3 causes an in- crease in TGF-2 protein synthesis that is associated with a decrease in the growth rate of these cells (9-11). The addition of antibodies against TGF- partially blocks the effects of 1,25(OH) 2 D 3 . Other mechanisms that may be involved in the regulation of cellular growth by 1,25(OH) 2 D 3 include changes in the expres- sion of immediate early genes such as c-myc, p21/WAF/ CIP1, alterations in protein kinase C activity and changes in polyamine synthesis (15-30,34). Apoptosis is altered in cells treated with the hormone (35-43). In order to more completely define the factors that might mediate the regulation of cellular growth and differen- tiation by 1,25(OH) 2 D 3 , we performed differential dis- play PCR in cells treated with the hormone. We found that the hormone decreased the expression of a novel early response gene, IEX-1 (44,45). The present report describes the regulation of the IEX-1 gene in keratino- cytes by cell density and by 1,25(OH) 2 D 3 . METHODS AND MATERIALS Culture of normal human keratinocytes. Human keratinocytes were isolated from neonatal foreskin specimens, and cell cultures were maintained in an undifferentiated, replicative state by growth and passage at sub-confluence in complete, serum-free MCDB 153 medium (10). Complete MCDB 153 medium with growth factors (GF+) includes 0.1 mM calcium and is supplemented with 0.2% (vol./vol.) bovine pituitary extract, epidermal growth factor (10 ng/ ml), and insulin (5 g/ml). Standard MCDB 153 medium without growth factors (GF-) is prepared by excluding bovine pituitary ex- tract, EGF and insulin from the culture medium. Culture of human fetal osteoblast cells. Human fetal osteoblasts were obtained from Dr. Thomas C. Spelsberg and were grown in DMEM F12 medium as described (47). Differential display PCR. This analysis was performed using the method of Liang and Pardee (48-51). 1 Address correspondence to: Dr. Mark Pittelkow, Mayo Clinic, 200 First, SW, Guggenheim 4, Rochester, MN 55905. Fax: 507-284-1086. E-mail: pittelkow.mark@mayo.edu or Dr. Rajiv Kumar, Mayo Clinic, 200 First St., SW, 911A Guggenheim Bldg., Rochester, MN 55905. Fax: 507-266-2710. E-mail: rkumar@mayo.edu. Supported by NIH Grant DK-25409 (RK), Dermatology Founda- tion Research Fellowship, 1997 (TK), and Mayo Foundation (RK, MRP). BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 251, 868 – 873 (1998) ARTICLE NO. RC989556 868 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.