The role of estrogen receptor-α and its activation function-1 for growth plate closure in female mice.

High estradiol levels in late puberty induce growth plate closure and thereby cessation of growth in humans. In mice, the growth plates do not fuse after sexual maturation, but old mice display reduced longitudinal bone growth and high-dose estradiol treatment induces growth plate closure. Estrogen receptor (ER)-α stimulates gene transcription via two activation functions (AFs), AF-1 and AF-2. To evaluate the role of ERα and its AF-1 for age-dependent reduction in longitudinal bone growth and growth plate closure, female mice with inactivation of ERα (ERα(-/-)) or ERαAF-1 (ERαAF-1(0)) were evaluated. Old (16- to 19-mo-old) female ERα(-/-) mice showed continued substantial longitudinal bone growth, resulting in longer bones (tibia: +8.3%, P < 0.01) associated with increased growth plate height (+18%, P < 0.05) compared with wild-type (WT) mice. In contrast, the longitudinal bone growth ceased in old ERαAF-1(0) mice (tibia: -4.9%, P < 0.01). Importantly, the proximal tibial growth plates were closed in all old ERαAF-1(0) mice while they were open in all WT mice. Growth plate closure was associated with a significantly altered balance between chondrocyte proliferation and apoptosis in the growth plate. In conclusion, old female ERα(-/-) mice display a prolonged and enhanced longitudinal bone growth associated with increased growth plate height, resembling the growth phenotype of patients with inactivating mutations in ERα or aromatase. In contrast, ERαAF-1 deletion results in a hyperactive ERα, altering the chondrocyte proliferation/apoptosis balance, leading to growth plate closure. This suggests that growth plate closure is induced by functions of ERα that do not require AF-1 and that ERαAF-1 opposes growth plate closure.

[Environmental lead poisoning from lead-glazed earthenware used for storing drinks]. / Intoxication environnementale par le plomb liée à la consommation de boisson conservée dans une cruche artisanale en céramique vernissée.

INTRODUCTION: Current unusual environmental sources of lead exposure mainly include traditional medicines, either ayurvedic remedies or others, traditional cosmetics (kohl, surma), and the use of traditional earthenware, for storage or cooking. CASE REPORTS: We report two cases of lead poisoning in adults initially identified by paroxysmal abdominal pain or anemia. In both cases, the environmental investigation evidenced one main source of lead exposure, namely a lead-glazed earthenware jug in which a drink was stored, "kefir" in the first case, and "kombucha" tea in the second one. CONCLUSION: It is recommended to search for lead intoxication in patients with unexplained anemia. Environmental sources of lead can be multiple. Their relative importance has to be ranked during the environmental investigation and among these, lead-glazed earthenware must be considered as a source of high lead exposure when drinks are stored inside and thus can soak.

Aberrant growth plate development in VDR/RXR gamma double null mutant mice.

VDR forms heterodimers with one of three RXRs, RXR alpha, RXR beta, and RXR gamma, and it is thought that RXR ligands can also modulate the trans-activation function of VDR/RXR heterodimers. In the present study we generated VDR/RXR gamma double null mutant mice to examine the convergent actions of vitamin D and vitamin A signaling and to explore the possibility of a functionally redundant VDR. Although RXR gamma(-/-) mice exhibited no overt abnormalities, VDR(-/-)/RXR gamma(-/-) mice appeared similar to VDR(-/-) mice, showing features typical of vitamin D-dependent rickets type II, including growth retardation, impaired bone formation, hypocalcemia, and alopecia. However, compared to VDR(-/-) mice, growth plate development in VDR(-/-)/RXR gamma(-/-) mutant mice was more severely impaired. Normalizing mineral ion homeostasis through dietary supplementation with high calcium and phosphorous effectively prevented rachitic abnormalities, except for disarranged growth plates in VDR(-/-)/RXR gamma(-/-) mutant mice, and alopecia in both VDR(-/-) and VDR(-/-)/RXR gamma(-/-) mutant mice. Histological analysis of VDR(-/-)/RXR gamma(-/-) growth plates revealed that development of the hypertrophic chondrocytes was selectively impaired. Thus, our findings indicated that the combined actions of VDR- and RXR gamma-mediated signals are essential for the normal development of growth plate chondrocytes, and raised the possibility that a functionally redundant VDR is present on chondrocytes as a heterodimer with RXR gamma.

NSD3, a new SET domain-containing gene, maps to 8p12 and is amplified in human breast cancer cell lines.

We describe the isolation and characterization of NSD3, the third member of a gene family including Nsd1 and NSD2. Murine Nsd1 was isolated in a search for proteins that interact with the ligand-binding domain of retinoic acid receptor alpha. NSD2 (also known as WHSC1 and MMSET) is located in the Wolf-Hirschhorn syndrome (WHS) critical region on 4p16.3 and is involved in multiple myeloma with t(4;14) translocations. The proteins Nsd1, NSD2, and NSD3 are highly similar within a block of about 700 amino acids. This block contains several conserved domains, such as the SET domain and the PHD finger, present in proteins involved in development and/or chromatin reorganization. The NSD3 gene consists of an 8.5-kb transcript composed of 23 coding exons and spans >90 kb of genomic DNA. NSD3 maps to chromosome band 8p12 and is amplified in several tumor cell lines and primary breast carcinomas.

Two novel RXR alpha isoforms from mouse testis.

We report the isolation from mouse testis cDNA of two novel RXR alpha isoforms, mRXR alpha 2 and mRXR alpha 3, with distinct sequences upstream of exon 2. These two isoforms encode a similar protein (mRXR alpha 2/3) which lacks that 28 N-terminal amino acid residues of the major RXR alpha isoform, mRXR alpha 1. The N-terminal activation function (AF-1) of mRXR alpha 2/3 appears altered when compared to that of mRXR alpha 1. mRXR alpha 2 and mRXR alpha 3 are specifically expressed in the testis, and their expression is strongly upregulated in this tissue at puberty. These observations increase the molecular complexity of RXRs, and indicate that RXR alpha may play a specific function during spermatogenesis.

Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene.

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two-dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.

Isolation of retinoic acid-repressed genes from P19 embryonal carcinoma cells.

Retinoic acid (RA) plays a critical role in normal development, growth and differentiation by modulating the expression of target genes. Using substractive hybridization cloning, we isolated two cDNAs, whose corresponding mRNAs are repressed upon RA treatment of P19 embryonal carcinoma (EC) cells. The cDNAs correspond to the serine hydroxymethyltransferase (shmt) gene and the early transposon, ETnMG1. RA appears to reduce the stability of ETnMG1 transcript. We also report the sequence of two different isoforms of mouse SHMT. Since SHMT activity is increased when cells are stimulated to proliferate and during the S phase of the cell cycle, we suggest that repression of shmt expression is an important step in RA-induced cell growth arrest and differentiation.

AP-2.2: a novel AP-2-related transcription factor induced by retinoic acid during differentiation of P19 embryonal carcinoma cells.

A 2.8-kb cDNA encoding a new transcription factor (AP-2.2) has been cloned from mouse P19 embryonal carcinoma cells, in which the corresponding mRNA begins to accumulate 30 min after retinoic acid (RA) addition. The predicted protein is 449 amino acids long and exhibits approximately 65% overall identity with other AP-2-related proteins (human AP-2, mouse AP-2alpha and beta). A 96-amino-acid-long sequence, which is almost fully conserved between all these proteins, corresponds to the previously characterized human AP-2 DNA binding domain. Expression of AP-2.2 in Escherichia coli generated a protein that formed a specific complex with the AP-2 recognition site GCCN3GGC. AP-2.2 activated transcription from a reporter gene containing an AP-2 DNA binding site and acted synergistically with RARalpha to activate transcription from the CRABPII gene promoter. Transcriptional activation required the AP-2.2 amino-terminal region that contains a domain rich in proline and glutamine residues. The pattern of AP-2.2 expression in adult tissues, which is distinct from that of AP-2alpha, is essentially restricted to male and female gonads, to most if not all the squamous epithelia, and to several exocrine glands.

Sequence and expression pattern of the Stra7 (Gbx-2) homeobox-containing gene induced by retinoic acid in P19 embryonal carcinoma cells.

The cDNA sequence of Stra7, a retinoic acid (RA)-inducible gene in P19 embryonal carcinoma (EC) cells, was determined. The deduced Stra7 protein contains a homeodomain highly similar to that of the previously described chicken CHox7 gene product, and is highly conserved during evolution, from hemichordates to vertebrates. The mouse Stra7 cDNA corresponds to the full-length form of the 77 bp homeodomain-encoding cDNA fragment which was previously cloned and termed MMoxA or Gbx-2. Reverse-transcriptase-PCR analysis revealed the presence of Stra7/Gbx-2 transcripts in the adult brain, spleen, and female genital tract, whereas no expression could be observed in heart, liver, lung, kidney, or testes. In situ hybridization analysis showed a restricted expression pattern of Stra7/Gbx-2 in the three primitive germ layers during gastrulation. Restricted expression was also detected in the pharyngeal arches. Subsequently, there were specific expression domains in the developing central nervous system, at the midbrain/hindbrain boundary and later in the cerebellum anlage, in certain rhombomeres, in dorsal regions of the spinal cord, and in the developing dorsal thalamus and corpus striatum.

Efficient cloning of cDNAs of retinoic acid-responsive genes in P19 embryonal carcinoma cells and characterization of a novel mouse gene, Stra1 (mouse LERK-2/Eplg2).

Pluripotent mouse P19 embryonal carcinoma (EC) cells have been extensively used as a developmental model system because they can differentiate in the presence of retinoic acid (RA) into derivatives of all three germ layers depending on RA dosage and culture conditions. The expression of several genes has been shown to be induced in RA-treated P19 EC cells and, interestingly, some of these genes may play important roles during mouse embryogenesis. In view of the increasing evidence that RA is a crucial signaling molecule during vertebrate development, we have initiated a study aimed at the systematic isolation of genes whose expression is induced in P19 cells at various times after exposure to RA. We describe here an efficient differential subtractive hybridization cloning strategy which was used to identify additional RA-responsive genes in P19 cells. Fifty different cDNA fragments corresponding to RA-induced genes were isolated. Ten cDNAs represent known genes, 4 of which have already been described as RA-inducible, while the remaining 40 correspond to novel genes. Many of these cDNA sequences represent low-abundance mRNAs. Kinetic analysis of mRNA accumulation following RA treatment allowed us to characterize four classes of RA-responsive genes. We also report the sequence and expression pattern in mouse embryos and adult tissues of one of these novel RA-inducible genes, Stra1, and show that it corresponds to the mouse ligand for the Cek5 receptor protein-tyrosine kinase.

The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18.

Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand-dependent activation function, AF-2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF-2 in yeast and interacts in a ligand-dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF-2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF-2 antagonist hydroxytamoxifen cannot promote ER-TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand-dependent AF-2. Interestingly, the TIF1 N-terminal moiety is fused to B-raf in the mouse oncoprotein T18.

Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.

We have previously reported that the binding site repertoires of heterodimers formed between retinoid X receptor (RXR) and either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) bound to response elements consisting of directly repeated PuG(G/T)TCA motifs spaced by 1-5 bp [direct repeat (DR) elements 1-5] are highly similar to those of their corresponding DNA binding domains (DBDs). We have now mapped the dimerization surfaces located in the DBDs of RXR, RAR and TR, which are responsible for cooperative interaction on DR4 (RXR and TR) and DR5 (RXR and RAR). The D-box of the C-terminal CII finger of RXR provides one of the surfaces which is specifically required for the formation of the heterodimerization interfaces on both DR4 and DR5. Heterodimerization with the RXR DBD on DR5 specifically requires the tip of the RAR CI finger as the complementary surface, while a 7 amino acid sequence encompassing the 'prefinger region', but not the TR CI finger, is specifically required for efficient dimerization of TR and RXR DBDs on DR4. Importantly, DBD swapping experiments demonstrate not only that the binding site repertoires of the full-length receptors are dictated by those of their DBDs, but also that the formation of distinct dimerization interfaces between the DBDs are the critical determinants for cooperative DNA binding of these receptors to specific DRs.

The mouse one P-domain (pS2) and two P-domain (mSP) genes exhibit distinct patterns of expression.

We have previously shown that the human pS2 gene, which codes for a secreted peptide of 60 amino acids, is expressed in a number of human carcinomas, including carcinomas of the breast, the pancreas, and the large bowel. Strong pS2 gene expression was also observed in the normal gastric mucosa and in the regenerative tissues surrounding ulcerous lesions of the gastrointestinal tract. A number of pS2 similar peptides, designated as P-domain peptides, have been described, notably the porcine (PSP), murine (mSP), and human (hSP) spasmolytic polypeptides, which correspond to duplicated pS2 proteins. We have now cloned a mouse homolog of the human pS2 cDNA to dispose of an animal model to study the pS2 protein function, which remains unknown at the present time. We show that the mouse putative pS2 protein sequence and the physiological pattern of expression of the mouse pS2 gene are well conserved. The mouse pS2 gene is highly expressed in the stomach mucosa cells, whereas no pS2 gene expression could be detected in the mouse mammary gland, even during postnatal development processes dependent on growth factors or hormones. Using in situ hybridization, we show that although coexpressed in the fundus, the antrum and the antrum-pyloric regions of the stomach, the mouse pS2 and mSP genes exhibit distinct and complementary cellular patterns of expression.

Identification of a novel retinoic acid receptor in regenerative tissues of the newt.

In urodele amphibians, the progenitor cells that regenerate amputated limbs (known as the blastema) normally replace only the missing structures. After systemic delivery of retinoic acid (RA), more proximal structures are also formed, indicating that RA can control position specification in the proximal-distal axis of the regenerating limb. According to dose and experimental context, retinoids can also re-specify the anteroposterior axis of the limb, induce deletions of skeletal elements, or block re-growth completely. To study the molecular basis of these morphogenetic effects, we screened complementary DNA libraries of newt regenerative tissues (limbs and tails) for hormone nuclear receptors activated by RA. Two functional retinoic acid receptors (RARs) were identified, one of which is the newt homologue of the human alpha-receptor (RAR alpha). The second receptor, called RAR delta, is novel. Sequence analysis suggests that the composite newt RAR previously reported is chimaeric, consisting of 5'RAR-beta-like and 3' RAR delta clones. We conclude that multiple RARs are expressed during limb regeneration in amphibians and suggest that receptor heterogeneity may underlie the different effects of retinoids on limb morphogenesis.

Identification of a second human retinoic acid receptor.

We have previously described a human complementary DNA that encodes a novel protein which is homologous to members of the steroid/thyroid nuclear receptor multigene family. This novel protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) which has been recently characterized. To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene (vit-tk-CAT) in the presence of possible receptor ligands. Here we show that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. This demonstrates the existence of two human retinoic acid receptors designated RAR-alpha and RAR-beta.

[The utilization of EB 51 in parenteral feeding. Clinical, biological and anatomopathological control. Statistical study on 30 patients]. / L'utilisation de l'eb 51 en alimentation parentérale. Contrôle clinique, biologique et anatomopathologique. Etude statistique sur 30 malades

Two groups of patients (comas of central origin and serious digestive undernourished) were submitted to an exclusive or a supplementary parenteral feeding for a short or a long period of time totaling over 500 days. The catheter was placed (in 80 p. cent of the cases) in a deep vein and was tunnellized. EB 51 (Trivemil) used in those 30 patients gives amino-acids, lipids and glucides. The caloric intake was completed by 30 p. cent glucose solutes. If the average caloric intake was 3.000 calories per day, it was often above that figure since the feeding was combined with a nutri-pump enteral feeding for 60 p. cent of the days. The clinical tolerance was quite good. On a biological level, in addition to classic investigations, 40 analytic graphic records of lipid levels were made, as well as a quantitative analysis of serous triglycerides and lipurias, a study of sorbitol and 500 nitrogen balances combined with 150 chromatographies on the acidaminuria column. An hepatic biological control was also made (amnoniemia, transaminases) as well as a quantitative analysis of lactates, pyruvates and minerals: iron-phosphorus-magnesium. Some of the results were checked by statistical studies. No serious anomaly was detected. Lung and hepatic biopsies carried out upon 9 patients did not apparently reveal any lipidic overloading. The clinical and the biological investigation show that this complete nutriment is perfectly tolerated and assimilated.