View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Keratinocyte G2/M Growth Arrest by 1,25-Dihydroxyvitamin D3 Is Caused by Cdc2 Phosphorylation Through Wee1 and Myt1 Regulation XiujuDai,KenshiYamasaki,LujunYang,KojiSayama,YujiShirakata,ShoTokumara,YokoYahata, Mikiko Tohyama, and Koji Hashimoto Department of Dermatology, Ehime University School of Medicine, Ehime, Japan 1,25-dihydroxyvitamin D3 (1,25[OH]2VD3) has an antiproliferative effect on keratinocyte growth, and its derivatives are used for the treatment of psoriasis. It was reported previously that 1,25[OH]2VD3 induced cell cycle arrest not only at the G0/G1 phase but also at the G2/M phase. However, the mechanism of 1,25[OH]2VD3-induced G2/M phase À8 À6 arrest in keratinocytes has not been fully understood. The addition of 10 to 10 M 1,25[OH]2VD3 to cultured À6 normal human keratinocytes enhanced the expression of Myt1 mRNA preceding Wee1 mRNA; 10 M 1,25[OH]2VD3 unregulated Myt1 mRNA from 6 h to 24 h and Wee1 mRNA from 12 to 48 h. Interestingly, the levels of phosphorylated Cdc2 were increased between 6 h and 48 h after 1,25[OH]2VD3 treatment, although the expression levels of Cdc2 mRNA and its protein production were reduced. 1,25[OH]2VD3 also decreased the expression of cyclin B1, which forms a complex with Cdc2. These data indicated that the increase of Myt1 and Wee1 induced the phosphorylation of Cdc2 leading to G2/M arrest. In conclusion, the induction of Cdc2 phosphorylation due to the increase of Wee1 and Myt1 as well as the reduction of Cdc2 and cyclin B1 are involved in 1,25[OH]2VD3-induced G2/M arrest of keratinocytes. Key words: 1,25-dihydroxyvitamin D3/Keratinocyte/Wee1/Myt1/Cdc2/cell cycle J Invest Dermatol 122:1356–1364, 2004 The compound 1,25-dihydroxyvitamin D3 (1,25[OH]2VD3), increased levels of p21 (Segaert et al, 1997; Zhuang and which is the hormonal form of vitamin D3, regulates cell Burnstein, 1998; Moffatt et al, 2001), p27 (Segaert et al, growth in a variety of cells (Walters, 1992). The skin is one of 1997; Wang et al, 1997), and transforming growth factor- the target organs for 1,25[OH]2VD3, and the vitamin D receptor bGF-b (Segaert et al, 1997), decreased Cdk2 activity (Wang (VDR) is present in the cells of both the epidermis and the et al, 1997; Zhuang and Burnstein, 1998), reduced levels of dermis (Stumpf et al, 1979, 1984; Pillai et al,1988;Mildeet al, cyclin E (Zhang et al, 1996), repressed E2F transactivation 1991). The effects of 1,25[OH]2VD3onkeratinocyteshave (Zhuang and Burnstein, 1998), and retinoblastoma hypo- been investigated extensively, and well characterized in vitro phosphorylation (Kobayashi et al, 1993; Segaert et al, 1997). and in vivo.Inthein vitro studies, 1,25[OH]2VD3 demonstrated Interestingly, 1,25[OH]2VD3 induces the accumulation of a potent inhibitory effect on proliferation, and promoted the cells not only in the G1 compartment but also in the G2/M differentiation of both murine and human keratinocytes compartment. These features have been noted in HL60 cell (Hosomi et al, 1983; Smith et al, 1986; Matsumoto et al, cultures (Godyn et al, 1994; Zhang et al, 1996; Harrison et al, 1990; Kobayashi et al, 1993; Gniadecki, 1996; Kobayashi et al, 1999), in breast cancer cell lines (Eisman et al, 1989a, b), 1998; Segaert et al, 2000). The critical effects on the epidermis and also in human keratinocytes (Kobayashi et al, 1993). In are also manifested in vivo (Holick et al, 1987; Dubertret et al, contrast to the numerous studies on the mechanism of G1 1992; el-Azhary et al, 1993; Langner et al, 1993). block in 1,25[OH]2VD3-treated keratinocytes, the underly- Several reports have demonstrated that 1,25[OH]2VD3 ing mechanism of G2 block induced by 1,25[OH]2VD3 in suppresses keratinocyte growth through cell cycle regula- keratinocytes has not been reported. tion. The most distinguished effect of 1,25[OH]2VD3 on cell Orderly progression through the cell cycle is mediated cycle regulation is G1 block. G1 block is associated with by the activation of a highly conserved family of protein changes of various kinds of cell cycle regulatory molecules: kinases, namely, cyclin-dependent kinases (Cdks) (Pines, 1995; Morgan, 1997). Activation of a particular Cdk requires binding to a specific regulatory subunit, which is termed Abbreviations: ATP, adenosine triphosphate; Cdc, cell division a cyclin. A pivotal regulatory step for G2/M transition in cycle; Cdk, cyclin-dependent kinase; 1,25[OH]2VD3, 1,25-dihy- eucaryotes is the activation of the cell division cycle (Cdc)2/ droxyvitamin D3; GAPDH, glyceraldehyde 3-phosphate dehydro- cyclin B complex (initially called the maturation or mitosis genase; RPA, ribonuclease protection assay; RXR, retinoid X receptor; Thr, threonine; Tyr, tyrosine; VDR, vitamin D receptor; promoting factor, MPF) (Coleman and Dunphy, 1994; VDRE, vitamin D response elements Morgan, 1997). Cdc2/cyclin B is maintained in an inactive Copyright r 2004 by The Society for Investigative Dermatology, Inc. 1356 122 : 6 JUNE 2004 1,25-DIHYDROXYVITAMIN D3 PHOSPHORYLATE CDC2 THROUGH WEE1 AND MYT1 INDUCTION 1357 form during the S and G2 phases by inhibitory phos- possibility that 1,25[OH]2VD3 induces G2/M cell cycle arrest phorylation of the Cdc2 residues threonine 14 (Thr14) in keratinocytes by affecting Cdc2/cyclin B1. and tyrosine 15 (Tyr15), which are positioned within the adenosine triphosphate (ATP)-binding cleft (Gould and Effect of 1,25[OH]2VD3 on Wee1 and Myt1 in human Nurse, 1989; Krek and Nigg, 1991; Jin et al, 1996). The keratinocytes The cDNA microarray analysis showed that inhibitory phosphorylation of Cdc2 is modulated by the 1,25[OH]2VD3 upregulated the expression of the Wee1 Wee1 family protein kinases, i.e., human Wee1 and Myt1. gene. To ascertain the effect of 1,25[OH]2VD3 on Wee1, we The Wee1 gene product is a tyrosine-specific protein examined the level of Wee1 mRNA, and Wee1 protein in kinase, which locates in nucleus and phosphorylates 1,25[OH]2VD3-treated human keratinocytes. Wee1 mRNA Cdc2 exclusively at Tyr15 (McGowan and Russell, 1993, expression was upregulated gradually in a time-dependent À6 1995). The Myt1 kinase, which is a homologue of the manner from 12 h up to 48 h by 10 M of 1,25[OH]2VD3 product of the mik1 gene, is a dual-specificity protein kinase (Fig 2A). The optimum induction of Wee1 mRNA was 6-fold that phosphorylates Cdc2 at both the Thr14 and Tyr15 at 48 h compared with the control (vehicle). Wee1 protein residues (Booher et al, 1997; Fattaey and Booher, 1997; began to increase at 12 h and reached the maximum, 3-fold Gould and Nurse, 1989; Krek and Nigg, 1991; Mueller et al, at 48 h (Fig 2B). This time course of Wee1 protein induc- 1995). Myt1 kinase is a membrane-associated protein that tion is consistent with that of Wee1 mRNA. Furthermore, localizes to the endoplasmic reticulum and Golgi complex 1,25[OH]2VD3 increased Wee1 mRNA expression in a (Liu et al, 1997); except for inhibitory phosphorylation of concentration-dependent manner, 1.6-fold at 10À8 M, 2.4- Cdc2, Myt1 seems to influence normal shuttling of the fold increase at 10À7M, and 4.0-fold 10À6 M (Fig 2C). Wee1 Cdc2/cyclin B complex into the nucleus (Liu et al, 1999). protein was increased almost similarly by the addition of À8 À7 À6 Phosphorylation of Tyr15 and Thr14 suppresses catalytic 1,25[OH]2VD3 at 10 ,10 , and 10 (Fig 2D). These data activity of Cdc2 by disrupting the orientation of the ATP demonstrate that 1,25[OH]2VD3 increases both Wee1 molecule to the ATP-binding cleft of Cdc2 (Atherton-Fessler mRNA and Wee1 protein similarly in human keratinocytes. et al, 1993; De Bondt et al, 1993). Cdc25 family members, Since Myt1 is a Wee1 family kinase that has been shown which are dual-specificity phosphatases, have been identi- to phosphorylate Cdc2 both at Tyr15 and Thr14 (Mueller fied as positive regulators of Cdc2. They counteract Wee1/ et al, 1995; Booher et al, 1997; Fattaey and Booher, 1997), Myt1 activities (Russell and Nurse, 1986; Sebastian et al, we examined the effect of 1,25[OH]2VD3 on Myt1 expres- 1993). Dephosphorylation of the Thr14 and Tyr15 residues sion. 1,25[OH]2VD3 enhanced the expression of Myt1 of Cdc2 by Cdc25B (in the cytoplasm) and Cdc25C (in the mRNA (Fig 3A). But the time course of Myt1 mRNA nucleus) in the late G2 phase activates the cyclin B/Cdc2 induction by 1,25[OH]2VD3 was distinguished from that of complex directly, and the accumulated, active Cdc2/ Wee1 mRNA. Myt1 mRNA increased markedly from 6 to cyclin B1 then triggers the initiation of mitosis (Dunphy 12 h, and decreased at 24 h. Then, Myt1 mRNA expres- and Kumagai, 1991; Strausfeld et al, 1991; Coleman and sion returned to the baseline level at 36 h. Myt1 protein Dunphy, 1994; Karlsson et al, 1999). Therefore, the active increased and decreased in the same time course as Myt1 À8 À7 status of the Cdc2/cyclin B complex is controlled by several mRNA (Fig 3B). 1,25[OH]2VD3 at 10 ,10, and G2/M-specific cell cycle regulatory kinases and phospha- 10À6 M increased Myt1 mRNA, 2.3-, 3-, and 1.5-fold, res- tases that reversibly phosphorylate Cdc2. pectively, compared with the control (vehicle) (Fig 3C). The To elucidate the mechanism of G2 block induced in optimum induction of Myt1 protein was also observed À7 human keratinocytes by 1,25[OH]2VD3, we investigated at 10 M of 1,25[OH]2VD3 (Fig 3D).