Regulation of the melanoma cell adhesion molecule gene in melanoma: modulation of mRNA synthesis by cyclic adenosine monophosphate, phorbol ester, and stem cell fFactor/c-kKit signaling.
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The melanoma cell adhesion molecule was identified as a human melanoma-associated antigen that increases in expression as tumors increase in thickness and begin to acquire metastatic potential. Clinical and experimental evidences suggest that the development of metastatic capacity might be the consequence of increased melanoma cell adhesion molecule expression. The mechanisms for upregulation of the melanoma cell adhesion molecule during melanoma progression are, however, still poorly understood. In this study, we show that melanoma cell adhesion molecule expression is tightly regulated at the transcriptional level. Using a combination of CAT reporter assays and semiquantitative reverse transcriptase-polymerase chain reaction, we observed that cyclic adenosine monophosphate significantly increases transcription of the melanoma cell adhesion molecule in nonmetastatic melanoma cells. In metastatic cells, transcription of the gene was constitutive and could not be further increased by cyclic adenosine monophosphate. On the other hand, melanoma cell adhesion molecule promoter activity was impeded upon treatment with phorbol esters or in the presence of stem cell factor, a phenomenon which was protein kinase C-dependent. Promoter-deletion studies demonstrated that the first 196 nt of the melanoma cell adhesion molecule promoter region are sufficient to get full expression in metastatic melanoma cells. This fragment contains five binding sites for the transcription factor Sp1 and DNA mobility shift experiments showed direct binding of Sp1 to the promoter. In conclusion, our results indicate that Sp1 is sufficient to drive constitutive melanoma cell adhesion molecule expression in metastatic melanoma cells. In nonmetastatic cells, however, melanoma cell adhesion molecule expression is repressed and we speculate that stem cell factor/c-Kit signaling might be responsible for the control of melanoma cell adhesion molecule synthesis, and thus, perhaps, of melanoma progression and metastasis. (+info)
Spatiotemporal expression pattern of keratins in skin of AP-2alpha-deficient mice.
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Transcription factor AP-2alpha has been implicated as being a cell-type-specific regulator of gene expression during vertebrate embryogenesis based on its expression pattern in neural crest cells, ectoderm, and the nervous system in mouse and frog embryos. In mice, AP-2alpha is expressed in surface ectoderm beginning at the single cell layer state around E8.75. AP-2alpha-deficient mice, derived by targeted mutagenesis, display a severe ventral closure defect resulting in cranio-abdominoschisis and a hypoplasia of the cranial ganglia. This study analyzed the effect of a targeted disruption of the AP-2alpha gene on the architecture and the expression of intermediate filaments in skin. We analyzed skin samples from newborn mice and found no difference in either the morphology of the skin or the amount of intermediate filaments expressed. This suggests that despite the results from other analyses, loss of transcription factor AP-2alpha does not affect the expression of intermediate filaments in the skin of newborn animals. We found an altered spatial distribution of intermediate filament expression in the single layered cranial ectoderm during days 9-12 of gestation leading to an evenly distributed expression of keratin 5 and 15 in the mutants. Furthermore, the mutants lack a ring of ectodermal cells highly positive for keratin 15 in the area where lens induction occurs, indicating a defect in the inductive interactions underlying eye formation. (+info)
Keratin 10 gene expression during differentiation of mouse epidermis requires transcription factors C/EBP and AP-2.
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The epidermis forms a vital barrier composed of stratified keratinocytes and their differentiated products. One of these products, keratin K10, is critical to epidermal integrity, because mutations in k10 lead to abnormal blistering. For the normal expression of k10, differentiation-associated transcription factors C/EBPalpha, C/EBPbeta, and AP-2 are well positioned to play an important role. Here, regulation of the k10 gene is examined in keratinocytes in the skin of normal mice and in transgenic mice carrying targeted deletions of c/ebpbeta and ap-2alpha. In cultured cells, C/EBPalpha and C/EBPbeta are each capable of activating the k10 promoter via three binding sites, identified by site-directed mutagenesis. In a given epidermal cell in vivo, however, the selection of C/EBPalpha versus C/EBPbeta for k10 regulation is determined via a third transcription factor, AP-2. This novel regulatory scheme involves: (1) unique gradients of expression for each transcription factor, i.e., C/EBPbeta and AP-2 most abundant in the lower epidermis, C/EBPalpha in the upper; (2) C/EBP-binding sites in the ap-2alpha gene promoter, through which C/EBPbeta stimulates ap-2alpha; and (3) AP-2 binding sites in the c/ebpalpha promoter, through which AP-2 represses c/ebpalpha. Promoter-analysis and gene-expression data presented herein support a regulatory model in which C/EBPbeta activates and maintains AP-2 expression in basal keratinocytes, whereas AP-2 represses C/EBPalpha in those cells. In response to differentiation signals, loss of AP-2 expression leads to derepression of the c/ebpalpha promoter and activation of k10 as cells migrate upward. (+info)
Auto-inhibition and partner proteins, core-binding factor beta (CBFbeta) and Ets-1, modulate DNA binding by CBFalpha2 (AML1).
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Core-binding factor alpha2 (CBFalpha2; otherwise known as AML1 or PEBP2alphaB) is a DNA-binding subunit in the family of core-binding factors (CBFs), heterodimeric transcription factors that play pivotal roles in multiple developmental processes in mammals, including hematopoiesis and bone development. The Runt domain in CBFalpha2 (amino acids 51 to 178) mediates DNA binding and heterodimerization with the non-DNA-binding CBFbeta subunit. Both the CBFbeta subunit and the DNA-binding protein Ets-1 stimulate DNA binding by the CBFalpha2 protein. Here we quantify and compare the extent of cooperativity between CBFalpha2, CBFbeta, and Ets-1. We also identify auto-inhibitory sequences within CBFalpha2 and sequences that modulate its interactions with CBFbeta and Ets-1. We show that sequences in the CBFalpha2 Runt domain and sequences C terminal to amino acid 214 inhibit DNA binding. Sequences C terminal to amino acid 214 also inhibit heterodimerization with the non-DNA-binding CBFbeta subunit, particularly heterodimerization off DNA. CBFbeta rescinds the intramolecular inhibition of CBFalpha2, stimulating DNA binding approximately 40-fold. In comparison, Ets-1 stimulates CBFalpha2 DNA binding 7- to 10-fold. Although the Runt domain alone is sufficient for heterodimerization with CBFbeta, sequences N terminal to amino acid 41 and between amino acids 190 and 214 are required for cooperative DNA binding with Ets-1. Cooperative DNA binding with Ets-1 is less pronounced with the CBFalpha2-CBFbeta heterodimer than with CBFalpha2 alone. These analyses demonstrate that CBFalpha2 is subject to both negative regulation by intramolecular interactions, and positive regulation by two alternative partnerships. (+info)
Purification and identification of a tissue-specific repressor involved in serum amyloid A1 gene expression.
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We have previously demonstrated that the 5'-flanking regions from the rat serum amyloid A1 (SAA1) promoter are necessary and sufficient to confer specific cytokine-induced expression in cultured hepatoma cells. Deletion analysis identified a tissue-specific repressor (TSR) regulatory element, located between bp -289 and -256, that functioned as a silencer, contributing to the transcription repression on SAA1 promoter in nonliver cells. When this 34-base pair TSR-binding element was used as a probe in electrophoretic mobility shift assays, an intense DNA-protein complex was detected in nuclear extracts from HeLa and several other nonliver tissues. This TSR binding activity, however, was undetectable in extracts from liver or liver-derived cells. The distribution of TSR binding activity is therefore consistent with its regulatory role in repressing SAA1 expression in a tissue-specific manner. In this study, we purified TSR protein from HeLa nuclear extracts and showed that it has a molecular mass of approximately 50 kDa. Surprisingly, protein sequencing and antibody supershift experiments identified TSR as transcription factor AP-2. Subsequent functional analysis showed that forced expression of AP-2 in HepG2 cells could indeed inhibit conditioned medium-induced SAA1 promoter activation. Moreover, expression of a dominant-negative mutant of AP-2 in HeLa cells or mutation of the AP-2-binding site led to derepression of the SAA1 promoter, presumably by neutralizing the inhibitory effects of the endogenous wild-type AP-2. Our results therefore demonstrate a novel function for AP-2 in the transcriptional repression of SAA1 promoter. Together with its tissue distribution, AP-2 may contribute to SAA1's highly liver-specific expression pattern by restricting its expression in nonliver cells. (+info)
Indispensable role of the transcription factor PEBP2/CBF in angiogenic activity of a murine endothelial cell MSS31.
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Mice lacking the AML1/PEBP2alphaB/CBFa2 gene or PEBP2beta/CBFb gene exhibit a defect in definitive hematopoiesis and die in utero because of hemorrhage in the central nervous system. Hematopoiesis in the embryo is considered to be tightly associated with vascular development. Here we examined whether PEBP2/CBF plays any role in angiogenesis besides that in definitive hematopoiesis. We found that AML1/PEBP2alphaB/CBFa2, PEBP2alphaA/CBFa1, and PEBP2beta/CBFb were expressed in a murine endothelial cell line MSS31. The expression of these molecules as well as the DNA binding activity of PEBP2/CBF were augmented by angiogenic growth factors such as bFGF and VEGF. Moreover, the expression of PEBP2 alpha/CBFa protein in endothelial cells was confirmed at the site of angiogenesis in vivo. To further clarify the role of PEBP2/CBF in angiogenesis, we established permanent transfectants of PEBP2 beta-MYH11 gene, one that interacts with the runt domain of the alpha subunit and deregulates PEBP2/CBF in a dominant interfering manner. Proliferation, migration, and tube formation of the PEBP2 beta-MYH11 transfectants were significantly reduced in comparison with those activities of the mock transfectants. These results suggest that transcription factor PEBP2/CBF plays an important role in angiogenesis. (+info)
AP-2 transcription factors in the regulation of ERBB2 gene transcription by oestrogen.
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Transcription of the ERBB2 oncogene is repressed by oestrogen in human breast cancer cells. We show that a 218 bp fragment of the human ERBB2 gene promoter is responsive to oestrogen in transient transfection in ZR75.1 and SKBR.3 cells when the oestrogen receptor is expressed. Deletion analysis of this fragment shows that a sequence located at the 5' end, which is known to mediate ERBB2 overexpression in breast cancer, is also responsible for the oestrogen response. This sequence binds AP-2 transcription factors and appears functionally identical to an element of the oestrogen-dependent enhancer described in the first intron of human ERBB2. We observed that oestrogen treatment down-regulates expression of AP-2 proteins but does not affect the DNA binding activity of AP-2. Constitutive expression of AP-2beta or AP-2gamma, but not AP-2alpha, abrogates the estrogenic repression. Our results demonstrate that AP-2 transcription factors are implicated in the oestrogenic regulation of ERBB2 gene expression and suggest a complex interplay involving the different AP-2 isoforms and other unidentified factors. (+info)
AP-2 family members regulate basal and cAMP-induced expression of human chorionic gonadotropin.
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The AP-2 family of transcriptional regulator proteins has three members, alpha, beta and gamma. AP-2alpha and gamma are expressed in placenta and in the human trophoblast cell line JEG-3. AP-2 has been shown to regulate expression of the placental human chorionic gonado-tropin (hCG) alpha- and beta-subunit genes, however, previous work did not distinguish between the family members. Tryptic peptides of the AP-2 protein complexes purified from JEG-3 cells by oligo-affinity chromatography using the hCGalpha AP-2 site match the amino acid sequence of AP-2gamma. The fact that AP-2gamma is present at significant levels and binds the hCGalpha trophoblast-specific element suggests that AP-2gamma is at least part of the binding complex in vivo and plays a role in regulating hCG expression. We show that mutation of each of four AP-2 binding sites within the hCGbeta promoter decreases expression in transfection assays, demonstrating that all four sites are required for maximal expression in JEG-3 cells. Furthermore, we find differences in regulation of the family members: AP-2alpha mRNA levels increase in response to cAMP while AP-2gamma mRNA levels do not. The demonstrated importance of the AP-2 sites in controlling hCGalpha and beta expression and the likely involvement of more than one family member suggest that a balance in AP-2 proteins is involved in coordinate regulation of these genes. Moreover, many placenta-restricted genes are regulated by AP-2 proteins, thus members of this family may play an important overall role in placenta-specific expression. (+info)