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EGF increases retinoid X receptor-α in human trophoblast cells in culture: Relationship with retinoic acid induced-hCG secretion
Placenta (1996), Vol. 17
A.2
HI~A-G and HLA-E and Maternal-PlacentalImmune Interactions. DAniel E. Gera~h~. Ni Lee, Akik9 Ishitani#, Hans Marquardt*, the ClinicalResearchDivision,Fred Hutchinson Cancer Research Center, 1124 ColumbiaSt., M374, Seattle, WA 98104-2092, #Nara Medical University, Nara 634, Japan, *BristoI-Nyers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121. A detailedanalysisof peptides hound to soluble and membrane HLAG proteins expressed in the B lymphoblastoid cell line 721.221 showed that like class Ia complexes, both the soluble and membrane forms of HLA-G consist of heavy and light chains complexed with nonamcric pcpddes in a 1:1:1 ratio. The two proteins bound essentially the same set of peptides which are derived from a variety of intracellular proteins and define a peptide motif for HLA-G. The peptidescontain Icucineat the carhoxyl terminus and proline or small hydrophobiaamino acids in position 3 followed I~yproline or glycine in position4. Further studies of peptides hound to HL.A-G extracted from placental tissue were perfon'ned using an HLA-G specific antibody. The results mirrored those from transfected cells, with two noteworthy differences. While the pcptide motif of HI..A-G is the same, the number of distinct peptides found in the placental derived HLA-G protein is substantially smalle~than that seen in transfectant desired HLA--G. Further, a single peptide species, apparently unique to placental tissue, accounted for about 15% of the popfide pool. Because we and others have found HLA-E coordinately expressed with I-ILA-Gand with classical class 1, we expanded these studies to include an analysis of peptides bound to HI..A-E. Surprisingly, we identifiedHLA-E bound pcptides which were derived from the leader sequance of other I-1LAclass I, suggesting that HLA-E expression is involvedin processing other I-R.Aclass I complexes, that other HLA class I direcdy control the functional expression of HLA-E, or both. Additional evidence was obtained that cell lines and placental tissue which express HLA-G but not classical class I also express FILA-E and that indeed, one of the I-ILA-E-boundi~ptides identified from one such cell linewas derivedfrom the HLA-G leader sequence. These results will be discussed in terms of their relevance to potential functions for HI.A-E and HLA-G in rive.
Distribution of Type I Interferon Receptors in H~.~, Trophoblast Tissues and Isolated Cells. L. P a u l e s u a, R. R o m a g n o l i a, and M.G. R i c c i =. I n s t i t u t e s of G e n e r a l P h y s i o l o g y a a n d O b s t e t r i c s a n d G y n e c o l o g y c, U n i v e r s i t y of Siena, Siena, Italy. Numerous recent publications have shown that type I IFN is secreted at the materno-fetal interface of different species. However, in contrast to ovine and bovine trophoblastic IFN, ~hich ks known to play a critical role in the recognition of pregnancy, its role in species different from ruminants is completely undefined. In an investigation of the potential role of this eytoklne in the hum=n placenta, we investigated the distribution of its specific receptors in placental tissues and in the c~otrophoblast isolated ceils. By using immunohistochemical analysis and monoclonal antibodies we found that the receptor was mainly expressed by the highly p~oliferative and invasive trophoblast cells either forming the internal layer of villi leytotrophoblast) or expanding from the taps of some villi (trophoblast cell columns). Cytotrophoblast cells i~ colture also expressed the receptor. our data indicate that type I IFN can be considered a potential regulator of ~rophoblast growth and invasion. Therefore a pure trophoblast cell preparation is a suitable mod~l to study the effect of this t~Fpe of IFN.
EGF INCREASES RETINOID X RECEPTOR-a IN HUMAN TROPHOBLAST CELLS IN CULTURE: RELATIONSHIP WITH RETINOIC ACID INDUCED -hCG SECRETION. S. Roulier, C. Rochette-Egly*, E. Alsat, J. Guibourdenche and D~ Evain-Brion. INSEI'CM 1J427, Facultd des Sciences Pharmaceutiques et Biologiques, 75006 Paris, a n d " IGBMC, BP 163, 67404"Illkirch 5trasbourg, France. In the present study, the effect of rethaoic acid (RA) and Epidermal Growth Factor (EGF) on hCG secretion by human term trophoblast cells in culture, was analysed. In these cells, in contrast to EGF, RA alone did not significantly stimulate hCG secretion. However, RA potentiated the increase ha hCG secretion induced by EGF. To gain a better understanding of such a synergistic effect, the expression of retinoic acid receptors (RARs and RXRs) was compared ha control and ha EGF-treated cells. EGF treatment specifically increased the level of RXRa protein and mRNA. In parallel, we demonstrated that the choriocarcinoma cells lEG 3, which respond to RA by an increase ha hCG secretion, express constitutively high levels of RXRa protein. Furthermore, R.XRa-transfected trophoblast cells also become RA responsive for hCG secretion. All these data suggest that RXRa expression is modulated by EGF, and may be involved ha the effect of ILk on hCG secretion.
The 4311 Spongiotrophoblast-Specific Enhancer is Controlled by Placental Factors Other than Mash-2. S. Tanaka, T. C a h o n e t t i , S. Austin, M. Ku, and J. Rossant, S a m u e l L u n e n f e l d Research Institute, M o u n t Sinai Hospital, Toronto, Ontario, C a n a d a M 5 G 1X5. To understand the molecular m e c h a n i s m s of trophoblast development, the sequence and transcriptional regulation of the mouse 4311 gene has been analyzed. 4311 is expressed in a subset of ectoplacental cone (EPC) cells and later in spongiotrophoblast (SP) cells until term, and has h o m o l o g y to cathepsin L. W e have recently shown that a 340-bp enhancer region linked to the minimal promoter o f 4 3 1 1 is sufficient for SP-specific expression of 8-galactosidase (Sgal) in transgenic mice. Mice with a mutation in the transcription factor Mash-2 lack the SP at ElO.5 and do not express 4311 in the EPC, s u g g e s t i n g that Mash-2 m i g h t play a role in the regulation of 4311 expression. The 4311 enhancer region contains three putative E-box sequences (El-3). Although E 2 s h o w e d high binding affinity with Mash-2/E47 d i m e r s in electrophoretic mobility shift assays (EMSA), Mash-2 did not activate CAT-reporter gene expression under the control of the 4311 enhancer and minimal promoter region in either cotransfected C3H 10T1/2 cells or Jag-3 cells. 4 3 1 1 L a c Z constructs containing a mutated E 2 still s h o w e d SP-specific expression o f 13gal in transgenic placentae. Moreover, expression of the 431 l L a c Z construct was detected at E7.5 in the E P C of Mash-2 mutant embryos. These results suggest that t:actors other than Mash-2 can direct 4311 expression via the 340bp enhancer region. An E M S A using E 12.5 placental nuclear extract s h o w e d that a 60-bp region within the enhancer was responsible for the formation o f s p e c i f i c c o m p l e x e s with placental nuclear proteins.
A.2
HI~A-G and HLA-E and Maternal-PlacentalImmune Interactions. DAniel E. Gera~h~. Ni Lee, Akik9 Ishitani#, Hans Marquardt*, the ClinicalResearchDivision,Fred Hutchinson Cancer Research Center, 1124 ColumbiaSt., M374, Seattle, WA 98104-2092, #Nara Medical University, Nara 634, Japan, *BristoI-Nyers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121. A detailedanalysisof peptides hound to soluble and membrane HLAG proteins expressed in the B lymphoblastoid cell line 721.221 showed that like class Ia complexes, both the soluble and membrane forms of HLA-G consist of heavy and light chains complexed with nonamcric pcpddes in a 1:1:1 ratio. The two proteins bound essentially the same set of peptides which are derived from a variety of intracellular proteins and define a peptide motif for HLA-G. The peptidescontain Icucineat the carhoxyl terminus and proline or small hydrophobiaamino acids in position 3 followed I~yproline or glycine in position4. Further studies of peptides hound to HL.A-G extracted from placental tissue were perfon'ned using an HLA-G specific antibody. The results mirrored those from transfected cells, with two noteworthy differences. While the pcptide motif of HI..A-G is the same, the number of distinct peptides found in the placental derived HLA-G protein is substantially smalle~than that seen in transfectant desired HLA--G. Further, a single peptide species, apparently unique to placental tissue, accounted for about 15% of the popfide pool. Because we and others have found HLA-E coordinately expressed with I-ILA-Gand with classical class 1, we expanded these studies to include an analysis of peptides bound to HI..A-E. Surprisingly, we identifiedHLA-E bound pcptides which were derived from the leader sequance of other I-1LAclass I, suggesting that HLA-E expression is involvedin processing other I-R.Aclass I complexes, that other HLA class I direcdy control the functional expression of HLA-E, or both. Additional evidence was obtained that cell lines and placental tissue which express HLA-G but not classical class I also express FILA-E and that indeed, one of the I-ILA-E-boundi~ptides identified from one such cell linewas derivedfrom the HLA-G leader sequence. These results will be discussed in terms of their relevance to potential functions for HI.A-E and HLA-G in rive.
Distribution of Type I Interferon Receptors in H~.~, Trophoblast Tissues and Isolated Cells. L. P a u l e s u a, R. R o m a g n o l i a, and M.G. R i c c i =. I n s t i t u t e s of G e n e r a l P h y s i o l o g y a a n d O b s t e t r i c s a n d G y n e c o l o g y c, U n i v e r s i t y of Siena, Siena, Italy. Numerous recent publications have shown that type I IFN is secreted at the materno-fetal interface of different species. However, in contrast to ovine and bovine trophoblastic IFN, ~hich ks known to play a critical role in the recognition of pregnancy, its role in species different from ruminants is completely undefined. In an investigation of the potential role of this eytoklne in the hum=n placenta, we investigated the distribution of its specific receptors in placental tissues and in the c~otrophoblast isolated ceils. By using immunohistochemical analysis and monoclonal antibodies we found that the receptor was mainly expressed by the highly p~oliferative and invasive trophoblast cells either forming the internal layer of villi leytotrophoblast) or expanding from the taps of some villi (trophoblast cell columns). Cytotrophoblast cells i~ colture also expressed the receptor. our data indicate that type I IFN can be considered a potential regulator of ~rophoblast growth and invasion. Therefore a pure trophoblast cell preparation is a suitable mod~l to study the effect of this t~Fpe of IFN.
EGF INCREASES RETINOID X RECEPTOR-a IN HUMAN TROPHOBLAST CELLS IN CULTURE: RELATIONSHIP WITH RETINOIC ACID INDUCED -hCG SECRETION. S. Roulier, C. Rochette-Egly*, E. Alsat, J. Guibourdenche and D~ Evain-Brion. INSEI'CM 1J427, Facultd des Sciences Pharmaceutiques et Biologiques, 75006 Paris, a n d " IGBMC, BP 163, 67404"Illkirch 5trasbourg, France. In the present study, the effect of rethaoic acid (RA) and Epidermal Growth Factor (EGF) on hCG secretion by human term trophoblast cells in culture, was analysed. In these cells, in contrast to EGF, RA alone did not significantly stimulate hCG secretion. However, RA potentiated the increase ha hCG secretion induced by EGF. To gain a better understanding of such a synergistic effect, the expression of retinoic acid receptors (RARs and RXRs) was compared ha control and ha EGF-treated cells. EGF treatment specifically increased the level of RXRa protein and mRNA. In parallel, we demonstrated that the choriocarcinoma cells lEG 3, which respond to RA by an increase ha hCG secretion, express constitutively high levels of RXRa protein. Furthermore, R.XRa-transfected trophoblast cells also become RA responsive for hCG secretion. All these data suggest that RXRa expression is modulated by EGF, and may be involved ha the effect of ILk on hCG secretion.
The 4311 Spongiotrophoblast-Specific Enhancer is Controlled by Placental Factors Other than Mash-2. S. Tanaka, T. C a h o n e t t i , S. Austin, M. Ku, and J. Rossant, S a m u e l L u n e n f e l d Research Institute, M o u n t Sinai Hospital, Toronto, Ontario, C a n a d a M 5 G 1X5. To understand the molecular m e c h a n i s m s of trophoblast development, the sequence and transcriptional regulation of the mouse 4311 gene has been analyzed. 4311 is expressed in a subset of ectoplacental cone (EPC) cells and later in spongiotrophoblast (SP) cells until term, and has h o m o l o g y to cathepsin L. W e have recently shown that a 340-bp enhancer region linked to the minimal promoter o f 4 3 1 1 is sufficient for SP-specific expression of 8-galactosidase (Sgal) in transgenic mice. Mice with a mutation in the transcription factor Mash-2 lack the SP at ElO.5 and do not express 4311 in the EPC, s u g g e s t i n g that Mash-2 m i g h t play a role in the regulation of 4311 expression. The 4311 enhancer region contains three putative E-box sequences (El-3). Although E 2 s h o w e d high binding affinity with Mash-2/E47 d i m e r s in electrophoretic mobility shift assays (EMSA), Mash-2 did not activate CAT-reporter gene expression under the control of the 4311 enhancer and minimal promoter region in either cotransfected C3H 10T1/2 cells or Jag-3 cells. 4 3 1 1 L a c Z constructs containing a mutated E 2 still s h o w e d SP-specific expression o f 13gal in transgenic placentae. Moreover, expression of the 431 l L a c Z construct was detected at E7.5 in the E P C of Mash-2 mutant embryos. These results suggest that t:actors other than Mash-2 can direct 4311 expression via the 340bp enhancer region. An E M S A using E 12.5 placental nuclear extract s h o w e d that a 60-bp region within the enhancer was responsible for the formation o f s p e c i f i c c o m p l e x e s with placental nuclear proteins.