Co-localization of KLF6 and KLF4 with pregnancy-specific glycoproteins during human placenta development Loïc Blanchon, José Luis Bocco, Denis Gallot, Anne-Marie Gachon, Didier Lémery, Pierre Déchelotte, Bernard Dastugue, Vincent Sapin

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Loïc Blanchon, José Luis Bocco, Denis Gallot, Anne-Marie Gachon, Didier Lémery, et al.. Co- localization of KLF6 and KLF4 with pregnancy-specific glycoproteins during human placenta de- velopment. Mechanisms of Development, Elsevier, 2001, 105 (1-2), pp.185 - 189. ￿10.1016/S0925- 4773(01)00391-4￿. ￿hal-01918622￿

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Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Mechanisms of Development 105 (2001) 185±189 www.elsevier.com/locate/modo Gene expression pattern Co-localization of KLF6 and KLF4 with pregnancy-speci®c glycoproteins during human placenta development

LoõÈc Blanchona, Jose Luis Boccob, Denis Gallotc, Anne-Marie Gachona, Didier LeÂmeryc, Pierre DeÂchelotted, Bernard Dastuguea, Vincent Sapina,*

aINSERM U.384, Faculte de MeÂdecine, 63000 Clermont-Ferrand, France bUniversidad Nacional de Cordoba, Cordoba, Argentina cUnite de MeÂdecine Materno-Foetale, Clermont-Ferrand, France dService d'Anatomo-Pathologie, Clermont-Ferrand, France Received 27 February 2001; received in revised form 17 April 2001; accepted 23 April 2001

Abstract Pregnancy-speci®c glycoproteins (PSGs) are major placental proteins essential for the maintenance of normal gestation. However, little is known about their gene expression regulation during placentation. It was previously demonstrated that the human core promoter binding protein recently renamed KruÈppel-like factor (KLF) 6 binds to a highly conserved sequence within the PSG promoters and is mainly expressed in human term placenta. Here, we determined the expression pattern of the 13 other KLFs during human placental development. We demonstrate that eight KLFs exhibit speci®c expression patterns in human placental tissues and membranes, in favor of a functional cooperation of speci®c KLFs during placentation. In addition, we demonstrate that KLF6, KLF4 and PSG proteins are co-expressed in same cell types of placental villi and membranes. This experimental evidence further strengthens the potential cross talk of both transcription factors for PSG gene regulation in vivo. q 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Development; Human placenta; Trophoblast; KruÈppel-like factor; Pregnancy-speci®c glycoprotein

The placenta is composed by the extraembryonic cyto- transcription factors (Turner and Crossley, 1999). The trophoblastic cells, which progressively differentiate into KLF molecules have been shown to exhibit important tissue multinucleated syncytiotrophoblasts (Kaufmann and speci®c developmental regulatory functions (Dang et al., Burton, 1994). The syncytiotrophoblasts synthesize a 2000). All KLFs could bind similar promoter sequences, number of pregnancy proteins such as the pregnancy-speci- present in several genes (Okano et al., 2000). Together, ®c glycoproteins (PSGs) (Cross et al., 1994). these observations are in agreement with cooperation The PSGs are the major placental proteins secreted to between speci®c KLFs aimed to orchestrate tissue-speci®c maternal circulation during pregnancy with still unresolved developmental gene expression. function (Khan et al., 1992). PSG biosynthesis is mainly In order to gain knowledge regarding the involvement of regulated at transcriptional level during human placental KLFs during placentation, we analyzed the expression of development (Bocco et al., 1989), implicating an in vitro the fourteen KLFs along with the PSG products during interaction of GC-box motif with KLF6, which could be different steps of human pregnancy. functional in vivo for regulation of PSG genes (Koritsch- oner et al., 1996, 1997; Slavin et al., 1999) KLF6 belongs to a new family of KruÈppel zinc ®nger 1. Results and discussion KLF1/EKLF (Miller and Bieker 1993), KLF5/BTEB2/ * Corresponding author. Tel.: 133-4-7360-8024; fax: 133-4-7327-6132. IKLF (Anderson et al., 1995), KLF9/BTEB (Imataka et al., E-mail address: [email protected] (V. Sapin). 1992), KLF12/AP2-rep (Imhof et al., 1999), KLF13 and Abbreviations: BKLF, basic KLF; BTEB, basic transcription element bind- KLF14/KKLF (Scohy et al., 2000) were never expressed at ing protein; CPBP, core promoter binding protein; EKLF, erythroid KLF; detectable levels in placental tissues. In contrast, the eight GKLF, gut KLF; IKLF, intestinal KLF; LKLF, lung KLF; KLF, KruÈppel- other KLFs showed a differential expression pattern in like factor; KKLF, kidney KLF; PLH, placental lactogen hormone; PSG, pregnancy-speci®c glycoprotein; TGF, transforming growth factor; TIEG, placental tissues and/or membranes (Table 1). TGFb immediate-early gene; UKLF, ubiquitous KLF. Showing a similar expression pattern than PSG tran-

0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0925-4773(01)00391-4 186 L. Blanchon et al. / Mechanisms of Development 105 (2001) 185±189 KLF1, -5, -9, -13, -14 KLF12 (AP-2rep) KLF11 (TIEG2) KLF10 (TIEG1) KLF8 (BKLF3) KLF7 (UKLF) KLF6 (CPBP) KLF4 (GKLF) KLF3 (BKLF) (LKLF) 122222 2 2 2 1122121212 1112121212 1111111212 1111111112 212222 2 1221121212 2 1111111212 1111111112 111112 2 1111111111 1111121111 PSG-5 KLF2 origin) BeWO and JAR JEG-3 14th week (second trimester) 29th week (third trimester) 37th week (term) Placental membranes Chorion (extra-embryonic Placental tissues 6th week (®rst trimester) Amnion (embryonic origin) Trophoblastic cells Primary culture of trophoblasts Cell lines: Table 1 Summary of the KLF gene expression pro®les during human placentation in villi and placental membranes L. Blanchon et al. / Mechanisms of Development 105 (2001) 185±189 187 scripts (Streydio and Vassart, 1990), four KLF members that KLF6 is also involved in the TGFb signaling pathway were expressed at all stages analyzed by reverse transcrip- (Kim et al., 1998; Kojima et al., 2000). tase±polymerase chain reaction (RT±PCR). As expected, Four KLF family members were not detected during the the KLF6 gene expression correlated directly with the ®rst early steps of pregnancy, namely KLF3/BKLF (Cross- PSG pattern, further supporting its function as regulator of ley et al., 1996), KLF4/GKLF (Shields et al., 1996), KLF7/ PSG genes transcription in vivo. UKLF (Matsumoto et al., 1998) and KLF10/TIEG1 (Cook The function assigned for the KLF2/LKLF molecule is et al. 1998). Nevertheless, the KLF4 expression became also of biological interest for placental development. This detectable and was co-expressed together with KLF6 in molecule is involved in the assembly of the vascular tunica the placenta from mid-gestation until parturition. Note that and blood vessel stabilization during development (Kuo et these proteins co activated the human keratin-4 promoter al., 1997), both of which are biological functions also neces- (Okano et al., 2000). sary for placental vasculogenesis (Cross et al., 1994). At term, human placenta expressed eight KLFs, with KLF11, also called TIEG-2, was initially identi®ed as a similar expression whether the placenta was obtained by TGFb-inducible immediate-early gene transcription factor chirurgical cesarean or spontaneous parturition, excluding (Cook et al., 1998). Taking into account that the TGFb implications of these KLFs for labor parturition. pathway plays an essential role for normal placentation, it As previously described (Chou and Zilberstein, 1990), is interesting to propose that KLF11 also mediates TGFb PSG is expressed in placental villi (Fig. 1C,I). Interestingly, signaling in this tissue. Additionally, it was demonstrated KLF6 was also detected in placental villi (Fig. 1D,F), but was

Fig. 1. Expression of KLF6, KLF4, PSG and PLH on human placental villi at term and in cytotrophoblasts. In situ mRNA detection encoding PSG (C), KLF6 (D) and KLF4 (E) proteins was done on adjacent slides. Immunodetection was performed on adjacent slides of placental villi and on primary cytotrophoblasts isolated from term placenta, for PSG (I,N), KLF6 (F,J,O), KLF4 (G,K,P), and PLH (H,L) proteins, respectively. Incubations with sense riboprobes (B) and primary antibodies free (M) were presented as negative controls. Histological organization of the placental villi at term is presented in (A) using methylene blue coloration. Magni®cation: A±H, £200; I±P, £400. EC, endothelial cells; FBV, fetal blood vessel; MB, maternal blood; PF, placental ®broblasts; PV, placental villi; St, syncytiotrophoblast. 188 L. Blanchon et al. / Mechanisms of Development 105 (2001) 185±189

Fig. 2. Expression of KLF6, KLF4 and PSG-5 proteins on human placental membranes. Immunodetection of PSG (C,F,K), KLF6 (D,G,L) and KLF4 (H) was performed on human amniotic (A±H) and chorionic (I±L) membranes at term. Primary antibody-free incubations (B,E,J) are presented as negative controls. Histological organization of the amniotic and chorionic membranes at term is presented in (A±I) using methylene blue coloration. Magni®cations: A±D and I, £200; E±H and J±L, £400. AE, amniotic epithelium; AM, amniotic mesoderm; CM, chorionic mesoderm; LFS, Langhans ®brinoid stria; SL, spongy layer. mainly co-expressed with PSG in syncytiotrophoblastic cells tory roles during erythropoiesis mainly through the control (Fig. 1I,J). The same expression pattern was found for KLF4 of globin gene expression (Dang et al., 2000). (Fig. 1E,G,K), strongly supporting the potential involvement KLF6, KLF4 and PSG molecules are also co-expressed in of this molecule in concert with KLF6 for the in vivo PSG amniotic (Fig. 2C,D,F,G,H and data not shown) as well as in gene regulation. This hypothesis is further strengthened by chorionic membranes (Fig. 2K,L and data not shown) at the presence of consensus sequence for KLF4/GKLF binding mRNAs and protein levels. in PSG promoter (Panzetta-Dutari et al., 2000). The KLF6, In conclusion, we demonstrate that human KLFs are KLF4 and PSG co-localization was still observed on primary speci®cally expressed in placenta throughout pregnancy. cultures of cytotrophoblasts cells (Fig. 1N±P). The KLF6 and KLF4 are co-expressed with PSG molecules We also analyzed KLF expression in the placental in placenta as well as in villi and membranes stressing their membranes at term where PSG biosynthesis was also cooperation for PSG gene expression control. reported (Plouzek and Chou, 1991). Expression of PSG mRNA was con®rmed in both, chorionic and amniotic membranes along with seven KLFs transcripts. Interest- 2. Methods ingly, the amnion and chorion membranes, which are from embryonic and extra-embryonic origin, respectively, In situ hybridization was performed as already described showed differences in the KLF expression pattern. The (Slavin et al. 1999). Primary culture of cytotrophoblasts was amnion and chorion membranes have been implicated in realized according to Jacquemin et al. (1998) and character- erythropoiesis during pregnancy (Moritz et al., 1997). It is ized using a speci®c marker, the cytokeratin 7 (Blaschitz et interesting to mention that both KLF2 and 3 share regula- al., 2000). The template cDNAs used to generate PSG, L. Blanchon et al. / Mechanisms of Development 105 (2001) 185±189 189

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