CITED1 Expression Research Articles

Identification of prospective factors promoting osteotropism in breast cancer: a potential role for CITED2

Breast cancer metastases develop in the bone more frequently than any other site and are a common cause of morbidity in the form of bone pain, pathological fractures, nerve compression and life-threatening hypercalcemia. Despite ongoing research efforts, the molecular and cellular mechanisms that regulate breast cancer cell homing to and colonization of the bone as well as resultant pathological bone alteration remain poorly understood. To identify key mediators promoting breast cancer metastasis to bone, we utilized an immunocompetent, syngeneic murine model of breast cancer metastasis employing the mammary tumor cell line NT2.5. Following intracardiac injection of NT2.5 cells in neu-N mice, metastases developed in the bone, liver and lung, closely mimicking the anatomical distribution of metastases in patients with breast cancer. Using an in vivo selection process, we established NT2.5 sublines demonstrating an enhanced ability to colonize the bone and liver. Genome-wide cDNA microarray analysis comparing gene expression between parental NT2.5 cells and established sublines revealed both known and novel mediators of bone metastasis and osteolysis, including the transcriptional co-activator CITED2. In further studies, we found that expression of CITED2 was elevated in human primary breast tumors and bone metastasis compared to normal mammary epithelium and was highest in breast cancer cell lines that cause osteolytic bone metastasis in animal models. In addition, reducing CITED2 expression in NT2.5 cells inhibited the establishment of bone metastasis and osteolysis in vivo, suggesting a potential role for CITED2 in promoting breast cancer bone metastasis.

Placental Insufficiency Associated with Loss of Cited1 Causes Renal Medullary Dysplasia

A number of studies have shown that placental insufficiency affects embryonic patterning of the kidney and leads to a decreased number of functioning nephrons in adulthood; however, there is circumstantial evidence that placental insufficiency may also affect renal medullary growth, which could account for cases of unexplained renal medullary dysplasia and for abnormalities in renal function among infants who had experienced intrauterine growth retardation. We observed that mice with late gestational placental insufficiency associated with genetic loss of Cited1 expression in the placenta had renal medullary dysplasia. This was not caused by lower urinary tract obstruction or by defects in branching of the ureteric bud during early nephrogenesis but was associated with decreased tissue oxygenation and increased apoptosis in the expanding renal medulla. Loss of placental Cited1 was required for Cited1 mutants to develop renal dysplasia, and this was not dependent on alterations in embryonic Cited1 expression. Taken together, these findings suggest that renal medullary dysplasia in Cited1 mutant mice is a direct consequence of decreased tissue oxygenation resulting from placental insufficiency.

Immunohistochemical and Molecular Characteristics of Follicular Patterned Thyroid Nodules with Incomplete Nuclear Features of Papillary Thyroid Carcinoma

Background : Follicular patterned thyroid nodules with incomplete nuclear features of papillary thyroid carcinoma (FTN-INPTCs) are difficult to diagnose, and their biological behavior and association with follicular variants of PTC (FVPTCs) have not yet been established. The aim of this study is to determine immunohistochemical and molecular characteristics of FTNINPTCs. Methods : We investigated immunohistochemical features (galectin-3, HBME-1, CK19, fibronectin-1, CITED1), BRAF V600E mutation and RASSF1A promoter methylation status in 30 FTN-INPTC cases, along with 26 FVPTCs, 21 follicular adenomas (FAs) and 14 nodular hyperplasias (NHs). Results : Expression of galectin-3, HBME-1, CK19 and CITED1 was significantly higher in FTN-INPTCs than in FAs or NHs, but expression of galectin-3, CK19 and fibronectin-1 was lower in FTN-INPTCs than in FVPTCs. The BRAF V600E mutation was not detected in the benign nodules or FTN-INPTCs, whereas 57% of FVPTCs had the mutation. RASSF1A promoter methylation was higher in FTN-INPTCs than in benign nodules but there was no difference between FTN-INPTCs and FVPTCs. Conclusions : Our results represent the borderline immunohistochemical and molecular characteristics of FTN-INPTC. We conclude that FTN-INPTC is an intermediate lesion between a benign nodule and a FVPTC, and that it is pathogenetically related to FVPTC.

Cited2 modulates hypoxia‐inducible factor–dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc

To determine whether nucleus pulposus cells of the intervertebral disc express hypoxia-inducible factor 2alpha (HIF-2alpha), and to assess the role of HIF-1 and HIF-2 in controlling cited2 and vascular endothelial growth factor (VEGF) expression. Rat cells were cultured under normoxic (21% O2) or hypoxic (2% O2) conditions, and expression and promoter activity of HIF-2 target genes were evaluated. Gain- or loss-of-function experiments were performed to investigate the contribution of HIF isoforms to cited2 activity as well as the role of cited2 in regulating VEGF expression. We found that HIF-2alpha protein was expressed in vivo and that protein and messenger RNA expression were similar under both normoxic and hypoxic conditions. However, there was a significant increase in HIF-2alpha transactivation under hypoxic conditions. With respect to functional activity, unlike the case in most other tissues, HIF-2 failed to increase the transcriptional activities of superoxide dismutase 2 and frataxin, 2 common target genes involved in radical dismutation. However, under hypoxic conditions, HIF-2 preferentially regulated the expression and promoter activity of cited2, a p300 binding protein. When HIF-2alpha or HIF-1alpha was suppressed, cited2 promoter activity was inhibited. Finally, we showed that forced expression or suppression of cited2 resulted in corresponding changes in expression of VEGF, a common target gene for HIF-1 and HIF-2 in the nucleus pulposus cells. Results of this study indicate that in nucleus pulposus cells, HIF-2 and HIF-1 modulate their own transcriptional activity through cited2. We suggest that the 2 arms of the regulatory circuit serve to maintain survival activities and inhibit angiogenesis in the healthy disc.

Epiblastic Cited2 deficiency results in cardiac phenotypic heterogeneity and provides a mechanism for haploinsufficiency

AimsDeletion of the transcription factor Cited2 causes penetrant and phenotypically heterogenous cardiovascular and laterality defects and adrenal agenesis. Heterozygous human CITED2 mutation is associated with congenital heart disease, suggesting haploinsufficiency. Cited2 functions partly via a Nodal→Pitx2c pathway controlling left–right patterning. In this present study we investigated the primary site of Cited2 function and mechanisms of haploinsufficiency.Methods and resultsA Cited2 conditional allele enabled its deletion in particular cell lineages in mouse development. A lacZ reporter cassette allowed indication of deletion. Congenic Cited2 heterozygous mice were used to investigate haploinsufficiency. Embryos were examined by magnetic resonance imaging, by sectioning and by quantitative real-time polymerase chain reaction (qRT-PCR). Epiblast-specific deletion of Cited2 using Sox2Cre recapitulated penetrant and phenotypically heterogenous cardiovascular and laterality defects. Neural crest-specific deletion using Wnt1Cre affected cranial ganglia but not cardiac development. Mesodermal deletion with Mesp1Cre resulted in low penetrance of septal defect. Mesodermal deletion with T-Cre resulted in adrenal agenesis, but infrequent cardiac septal and laterality defects. β-Galatactosidase staining and qRT-PCR demonstrated the efficiency and location of Cited2 deletion. Murine Cited2 heterozygosity is itself associated with cardiac malformation, with three of 45 embryos showing ventricular septal defect. Cited2 gene expression in E13.5 hearts was reduced 2.13-fold in Cited2+/− compared with wild-type (P = 2.62 × 10−6). The Cited2 target gene Pitx2c was reduced 1.5-fold in Cited2+/− (P = 0.038) hearts compared with wild-type, and reduced 4.9-fold in Cited2−/− hearts (P = 0.00031). Pitx2c levels were reduced two-fold (P = 0.009) in Cited2+/− embryos, in comparison with wild-type. Cited2 and Pitx2c expression were strongly correlated in wild-type and Cited2+/− hearts (Pearson rank correlation = 0.68, P = 0.0009). Cited2 expression was reduced 7474-fold in Sox2Cre deleted hearts compared with controls (P = 0.00017) and Pitx2c was reduced 3.1-fold (P = 0.013). Deletion of Cited2 with Mesp1Cre resulted in a 130-fold reduction in cardiac Cited2 expression compared with control (P = 0.0002), but Pitx2c expression was not affected.ConclusionThese results indicate that phenotypically heterogenous and penetrant cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left–right patterning, with a secondary cell-autonomous role in the mesoderm. Cardiac malformation associated with Cited2 haploinsufficiency may occur by reducing expression of key Cited2 targets such as Pitx2c.

Group interpersonal therapy reduces depression in adolescent survivors of war

<h3>Abstract</h3> Macrophages are highly dynamic innate immune cells that adopt temporary phenotypes, known as polarization states, in response to changing environmental signals to combat microbial infection and contribute to the...

CBP/p300-Interacting Protein CITED1 Modulates Parathyroid Hormone Regulation of Osteoblastic Differentiation

PTH regulates osteoblastic differentiation and activity and exerts different overall skeletal effects in vivo, depending on the schedule and dose of administration. In clonal Wt9 murine osteoblastic cells, mRNA and protein levels of CITED1 transcriptional coactivator were strongly up-regulated by human (h) PTH(1-34). Stimulation of CITED1 mRNA by PTH was transient, peaking at 4 h, concentration dependent, and blocked by actinomycin D but not cycloheximide. The stimulation was mimicked by forskolin, phorbol ester, and the cAMP-selective PTH analog [G(1),R(19)] hPTH (1-28) and inhibited completely by the protein kinase A inhibitor, H89 and partially by phorbol ester-induced protein kinase C depletion. Increased CITED1 expression was not maintained during persistent (24 h) PTH exposure. Cultured primary calvarial osteoblasts from neonatal homozygous or hemizygous CITED1-knockout (KO) mice achieved 2-fold greater mineralized nodule formation in comparison with wild type (WT) osteoblasts. This effect was blocked by restoration of CITED1 expression via adenoviral gene transfer. Intermittent administration of hPTH(1-34) (10 nm, for 4 h every 48 h) for 3-6 wk increased mineralization up to 2-fold over basal levels in both WT and CITED1 KO mouse calvarial cell cultures. Whereas the cAMP-selective [G(1),R(19)]hPTH(1-28) analog [at 100 nm, equivalent to 10 nm hPTH(1-34)] did not stimulate mineralization in WT cultures, it was twice as effective as hPTH(1-34) in CITED1 KO cultures. Thus, CITED1 negatively regulates osteoblastic differentiation in vitro and inhibits the cAMP-dependent stimulation of differentiation by intermittent PTH. We conclude also that PTH receptor signaling pathways independent of cAMP restrain osteoblastic differentiation, an effect normally obscured in the presence of CITED1 but revealed in its absence.

CITED2 mediates the paradoxical responses of HIF-1α to proteasome inhibition

Hypoxia-inducible factor-1alpha (HIF-1alpha) is destabilized via the ubiquitin-proteasome system. Thus HIF-1alpha expression is robustly upregulated by proteasome inhibition, but paradoxically its activity is reduced. In the present study, we investigated the mechanism underlying the paradoxical response of HIF-1alpha to proteasome inhibition. In both Hep3B and HEK293 cells, a proteasome inhibitor MG132 noticeably attenuated hypoxic induction of erythropoietin and VEGF mRNAs. MG132 inactivated HIF-1alpha C-terminal transactivation domain (CAD), independently of factor inhibiting HIF-1 (FIH) and inhibited p300 recruitment by HIF-1alpha. We next tested the possibility that CITED2 is involved in the HIF-1 inactivation. CITED2 was found to be degraded via the ubiquitin-proteasome system and thus was stabilized by proteasome inhibition. Both the activity and the p300 binding of HIF-1alpha were inhibited by CITED2 expression and recovered by CITED2 siRNA in the presence of MG132. These results suggest that CITED2 is stabilized by proteasome inhibition and inactivates HIF-1 by interfering with the HIF-1alpha-p300 interaction. This may be an important mode-of-action for proteasome inhibition-based cancer therapy.

Cited1 and Cited2 are differentially expressed in the developing kidney but are not required for nephrogenesis

Early kidney development in mammals is characterized by reciprocal tissue interaction between the ureteric bud and the metanephric mesenchyme. The coordinated response to this interaction is regulated largely at the transcriptional level. Here, we investigate the expression and function of Cited1, a transcriptional cofactor that we have previously implicated in kidney development. We show that Cited1 is expressed in the metanephric mesenchyme after invasion of the ureteric bud and that its expression is limited to the cap mesenchyme, those cells that aggregate most tightly around the tip of the ureteric bud and give rise to nephronic epithelium of the adult kidney. Cited1 is down-regulated during the initial stages of epithelial conversion and is not expressed past this progenitor stage. Despite its unique expression pattern, deletion of Cited1 does not disrupt kidney development. We hypothesized that this finding was due to functional redundancy with other members of this gene family. The expression pattern of Cited2 overlaps that of Cited1, but its deletion, either alone or in combination with Cited1, does not disrupt epithelial differentiation of the metanephric mesenchyme. From these studies, we conclude that Cited1 and 2 are dynamically expressed during kidney development, but are not required for nephrogenesis.

CITED1 Expression in Wilms' Tumor and Embryonic Kidney

Wilms' tumors, or nephroblastomas, are thought to arise from abnormal postnatal retention and dysregulated differentiation of nephrogenic progenitor cells that originate as a condensed metanephric mesenchyme within embryonic kidneys. We have previously shown that the transcriptional regulator CITED1 (CBP/p300-interacting transactivators with glutamic acid [E]/aspartic acid [D]-rich C-terminal domain) is expressed exclusively in these nephrogenic progenitor cells and is downregulated as they differentiate to form nephronic epithelia. In the current study, we show that CITED1 expression persists in blastemal cell populations of both experimental rat nephroblastomas and human Wilms' tumors, and that primary human Wilms' tumors presenting with disseminated disease show the highest level of CITED1 expression. Unlike the predominantly cytoplasmic subcellular localization of CITED1 in the normal developing kidney, CITED1 is clearly detectable in the nuclear compartment of Wilms' tumor blastema. These findings indicate that CITED1 is a marker of primitive blastema in Wilms' tumors and suggest that persistent expression and/or altered subcellular localization of CITED1 in the condensed metanephric mesenchyme could play a role in Wilms' tumor initiation and pathogenesis.

Regulation of Cited2 expression provides a functional link between translational and transcriptional responses during hypoxia

Background and purpose Protein synthesis rates are greatly reduced under hypoxic conditions as a consequence of an overall inhibition of mRNA translation. Certain specific mRNA species have the ability to escape this general translational repression. At the cellular level this results in differential protein expression during hypoxic conditions. The objective of this study was to characterize the translational regulation of the postulated HIF-1α antagonist Cited2. Materials and methods DU145 prostate carcinoma cells and mouse embryonic fibroblasts with a homozygous knock-in mutation for eIF2α (S51A) or wild-type eIF2α were exposed to severe hypoxia after which both total mRNA and efficiently translated mRNA were isolated. Quantitative RT-PCR was used to measure and compare changes in transcription (total mRNA) with changes in translation (efficiently translated mRNA fraction). Results We show using HIF-1α null MEF cells that transcriptional induction of Cited2 during hypoxia is dependent on HIF-1α. Although global mRNA translation is inhibited during hypoxia Cited2 mRNA remains efficiently translated. An evolutionary conserved upstream open reading frame (uORF) in the 5′UTR of Cited2 did not stimulate translation in an eIF2α dependent manner during hypoxia. Conclusions Selective translation Cited2 by an eIF2α independent mechanism establishes a link between translation and HIF-1 dependent transcription during hypoxia.

ERα–CITED1 co-regulated genes expressed during pubertal mammary gland development: implications for breast cancer prognosis

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.

Differential regulation of Foxo3a target genes in erythropoiesis.

The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5. Both Foxo3a and Stat5 promote erythroid differentiation. To understand the interplay of SCF and Epo in maintaining the balance between renewal and differentiation during erythroid development, we investigated differential Foxo3a target regulation by Epo and SCF. Expression profiling revealed that a subset of Foxo3a targets was not inhibited but was activated by Epo. One of these genes was Cited2. Transcriptional control of Epo/Foxo3a-induced Cited2 was studied and compared with that of the Epo-repressed Foxo3a target Btg1. We show that in response to Epo, the allegedly growth-inhibitory factor Foxo3a associates with the allegedly growth-stimulatory factor Stat5 in the nucleus, which is required for Epo-induced Cited2 expression. In contrast, Btg1 expression is controlled by the cooperation of Foxo3a with cyclic AMP- and Jun kinase-dependent Creb family members. Thus, Foxo3a not only is an effector of PKB but also integrates distinct signals to regulate gene expression in erythropoiesis.

CITED2 expression and its regulation in human adrenocortical cancer

CITED2, a CBP/p300 interacting transactivator, has been shown to be essential for murine adrenal development. Its overexpression leads to enhanced proliferation in different cell models and to oncogenic cell transformation.

CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor

CITED2 gene deletion in mice leads to adrenal agenesis. Therefore, we analyzed CITED2, a CBP/p300 interacting transactivator with transforming activity, in the human adrenal gland. In this study, we examined CITED2 expression in human embryonic and adult adrenal glands as well as adrenocortical carcinomas. As ACTH and basic fibroblast growth factor (bFGF) are connected to the physiology and growth of adrenocortical cells we studied the regulation of CITED2 by these factors in the NCI-H295R adrenocortical carcinoma cell line. We found CITED2 expression in the adult adrenal cortex as well in adrenocortical carcinomas. At an early stage of human adrenal organogenesis CITED2 could be located to the definitive zone of the developing adrenal gland using immunohistochemistry. In NCI-H295R cells, stimulation by bFGF led to a dose-dependent increase in CITED2 promotor activity, mRNA and protein expression while ACTH had no significant effect. The stimulatory effect of bFGF could be reduced by blocking mitogen-activated protein kinase activity using the MAPkinase kinase (MEK1)-inhibitor PD98059. CITED2 is expressed in embryonic and adult human adrenal glands as well as in adrenocortical cancer. It is connected to the signaling cascades of bFGF and its expression is modulated by mitogen-activated protein kinases. This suggests a novel role for CITED2 in human adrenal growth and possibly in adrenal tumorigenesis.

Expression of the Transcriptional Coactivator CITED1 in the Adult and Developing Murine Brain

The transcription coactivator CITED1 is an important mediator of transcriptional events regulated by estrogen or TGF-β. We used in situ hybridization to delineate the distribution of CITED1 mRNA in the adult and developing murine brain and found robust CITED1 expression in ventral hypothalamus and midbrain raphe. The distribution of CITED1 in these regions overlapped the reported expression of estrogen receptors α and β. Less intense expression of CITED1 was also evident in medial preoptic area, subfornical organ, thalamus and cerebral cortex. CITED1 mRNA in the arcuate nucleus (an area of active transcriptional modulation by TGF-β) was evident in postmigratory neurons as early as embryonic day 16. Expression of CITED1 in arcuate continued throughout postnatal development. CITED1 in developing cerebellum was first evident in external granule cells and was transiently expressed in the Purkinje cell/granule cell layer in a temporal pattern similar to estrogen receptor-β. The spatial and temporal distribution of CITED1 mRNA reported here is consistent with a role for CITED1 in the modulation of transcriptional events mediated by steroid hormone and cytokine signaling pathways.

Expression of CITED2 in human adrenal tissues

Expression of CITED2 in human adrenal tissues

Cited2is required both for heart morphogenesis and establishment of the left-right axis in mouse development

Establishment of the left-right axis is a fundamental process of vertebrate embryogenesis. Failure to develop left-right asymmetry leads to incorrect positioning and morphogenesis of numerous internal organs, and is proposed to underlie the etiology of several common cardiac malformations. The transcriptional modulator Cited2 is essential for embryonic development: Cited2-null embryos die during gestation with profound developmental abnormalities, including cardiac malformations, exencephaly and adrenal agenesis. Cited2 is also required for normal establishment of the left-right axis; we demonstrate that abnormal heart looping and right atrial and pulmonary isomerism are consistent features of the left-right-patterning defect. We show by gene expression analysis that Cited2 acts upstream of Nodal, Lefty2 and Pitx2 in the lateral mesoderm, and of Lefty1 in the presumptive floor plate. Although abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos, laterality defects are only observed in a proportion of these embryos. We have therefore used a combination of high-resolution imaging and three-dimensional (3D) modeling to systematically document the full spectrum of Cited2-associated cardiac defects. Previous studies have focused on the role of Cited2 in cardiac neural crest cell development, as Cited2 can bind the transcription factor Tfap2, and thus affect the expression of Erbb3 in neural crest cells. However, we have identified Cited2-associated cardiac defects that cannot be explained by laterality or neural crest abnormalities. In particular, muscular ventricular septal defects and reduced cell density in the atrioventricular (AV) endocardial cushions are evident in Cited2-null embryos. As we found that Cited2 expression tightly correlated with these sites, we believe that Cited2 plays a direct role in development of the AV canal and cardiac septa. We therefore propose that, in addition to the previously described reduction of cardiac neural crest cells, two other distinct mechanisms contribute to the spectrum of complex cardiac defects in Cited2-null mice; disruption of normal left-right patterning and direct loss of Cited2 expression in cardiac tissues.

CITED4 inhibits hypoxia-activated transcription in cancer cells, and its cytoplasmic location in breast cancer is associated with elevated expression of tumor cell hypoxia-inducible factor 1alpha.

The interaction of hypoxia-inducible factor 1alpha and the CH1 domain of the transcriptional coactivator p300/CBP is necessary for the expression of hypoxia responsive genes and tumor angiogenesis. The transcription factor CITED2 binds p300/CBP at the CH1 domain and functions as a negative regulator of hypoxia signaling by competing with hypoxia-inducible factor 1alpha. CITED4, a recently identified member of the CITED family, binds p300/CBP via the CH1 domain and functions as a coactivator for transcription factor AP-2. Here, we show that CITED4 blocks the binding of hypoxia-inducible factor 1alpha to p300 in vitro and inhibits hypoxia-inducible factor-1alpha transactivation and hypoxia-mediated reporter gene activation. These studies suggest that CITED4 might function as an inhibitor of hypoxia-inducible factor 1alpha. To explore the function of CITED4 in breast cancer, we determined its expression in normal, in situ and invasive breast cancers. We also correlated its expression in 286 invasive breast tumors with clinicopathological, hypoxia markers and survival. In contrast to the nuclear localization of CITED4 in normal breast tissue, breast tumors were characterized by cytoplasmic and nuclear localization. Nuclear CITED4 expression was significantly inversely associated with tumor hypoxia-inducible factor 1alpha (P < 0.05), tumor size (P = 0.03), tumor grade (P = 0.0001), and Chalkley vessel count (P = 0.04). CITED4 showed no significant correlation with patient age (P = 0.45), estrogen receptor (P = 0.11), or epidermal growth factor receptor (P = 0.48). These results show that breast cancer development is characterized by either nuclear loss or cytoplasmic translocation of CITED4, with consequent loss of hypoxia-inducible factor-1alpha transcriptional antagonist activity. This may be an important mechanism by which tumors enhance hypoxia-inducible factor expression and result in an aggressive phenotype.

016.Role of cited genes in placental morphogenesis: studies in null mutant mice

Cited1 and Cited2 interact with CBP and p300. CBP/p300 bind numerous proteins and evidence exists, for Cited2 at least, that Cited binding prevents the binding of other proteins to CBP/p300. Since CBP/p300 interact with many proteins, can acetylate protein and DNA, and act as a ubiquitin ligase, it is likely that Cited1 and Cited2 function at a number of sites during development. We have generated mice that carry a null mutant allele for each of these genes. Analysis of null mutant embryos demonstrates that both Cited1 and Cited2 are required for normal embryonic development and survival. Although both Cited1 and Cited2 are expressed in the developing embryo and placenta, it appears that abnormal placental development and function is the cause of embryonic death. The defect that develops in the placentas of Cited1 null mutants is not apparent until late in gestation (16.5dpc). Cited1 null mutants are smaller than controls at birth and die during the early postnatal period. The placentas of these mutants are disorganised, with spongiotrophoblasts projecting in to the labyrinthine layer. In addition, resin casts of the maternal blood spaces within these placentas revealed extremely enlarged blood sinuses. We are searching for factors that could result in the increased size of the maternal blood sinuses. Cited2 null placentas and embryos are significantly smaller than controls; mutants die 3/4 the way through gestation (15.5dpc). The null mutant placentas have proportionally fewer spongiotrophoblasts, trophoblast giant cells and invasive trophoblasts. In addition, resin casts of fetal vasculature of the placenta reveal that the capillary network is underdeveloped. Through the isolation of trophoblast stem (TS) cells we are exploring the possibility that TS cell proliferation and/or differentiation is impaired due to a lack of Cited2. We suspect that the development of the phenotype may relate to the Hypoxia Inducible Factor-1a (HIF1a) transcription factor as Cited2 expression is induced by HIF1 and it acts to negatively regulate its activity.