The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

The interactions between fibroblast growth factors (FGF) and their receptors have important roles in mediating mesenchymal-epithelial cell interactions during embryogenesis. In particular, Fgf10 is predicted to function as a regulator of brain, lung and limb development on the basis of its spatiotemporal expression pattern in the developing embryo. To define the role of Fgf10, we generated Fgf10-deficient mice. Fgf10-/- mice died at birth due to the lack of lung development. Trachea was formed, but subsequent pulmonary branching morphogenesis was disrupted. In addition, mutant mice had complete truncation of the fore- and hindlimbs. In Fgf10-/- embryos, limb bud formation was initiated but outgrowth of the limb buds did not occur; however, formation of the clavicles was not affected. Analysis of the expression of marker genes in the mutant limb buds indicated that the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) did not form. Thus, we show here that Fgf10 serves as an essential regulator of lung and limb formation.

MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, involved in diverse physiological and pathological processes. Although miRNAs can function as both tumour suppressors and oncogenes in tumour development, a widespread downregulation of miRNAs is commonly observed in human cancers and promotes cellular transformation and tumorigenesis. This indicates an inherent significance of small RNAs in tumour suppression. However, the connection between tumour suppressor networks and miRNA biogenesis machineries has not been investigated in depth. Here we show that a central tumour suppressor, p53, enhances the post-transcriptional maturation of several miRNAs with growth-suppressive function, including miR-16-1, miR-143 and miR-145, in response to DNA damage. In HCT116 cells and human diploid fibroblasts, p53 interacts with the Drosha processing complex through the association with DEAD-box RNA helicase p68 (also known as DDX5) and facilitates the processing of primary miRNAs to precursor miRNAs. We also found that transcriptionally inactive p53 mutants interfere with a functional assembly between Drosha complex and p68, leading to attenuation of miRNA processing activity. These findings suggest that transcription-independent modulation of miRNA biogenesis is intrinsically embedded in a tumour suppressive program governed by p53. Our study reveals a previously unrecognized function of p53 in miRNA processing, which may underlie key aspects of cancer biology.

Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.

1 alpha,25-Dihydroxyvitamin D3[1 alpha,25(OH)2D3], an active form of vitamin D, has roles in many biological phenomena such as calcium homeostasis and bone formation, which are thought to be mediated by the 1 alpha,25(OH)2D3 receptor (VDR), a member of the nuclear hormone receptor superfamily. However, the molecular basis for the actions of 1 alpha,25(OH)2D3 in bone formation, its role during development and VDR genetic polymorphisms for predicting bone mineral density are uncertain. To investigate the functional role of VDR, we generated mice deficient in VDR by gene targeting. We report here that in VDR null mutant mice, no defects in development and growth were observed before weaning, irrespective of reduced expression of vitamin D target genes. After weaning, however, mutants failed to thrive, with appearance of alopoecia, hypocalcaemia and infertility, and bone formation was severely impaired as a typical feature of vitamin D-dependent rickets type II (refs 8, 9). Unlike humans with this disease, most of the null mutant mice died within 15 weeks after birth, and uterine hypoplasia with impaired folliculogenesis was found in female reproductive organs. These defects, such as alopoecia and uterine hypoplasia, were not observed in vitamin D-deficient animals. The findings establish a critical role for VDR in growth, bone formation and female reproduction in the post-weaning stage.

Osteoclasts are thought to be solely responsible for the removal of bone matrix. However, we show here that osteocytes can also remove bone matrix by reversibly remodeling their perilacunar/canalicular matrix during the reproductive cycle. In contrast, no osteocytic remodeling was observed with experimental unloading despite similar degrees of bone loss. Gene array analysis of osteocytes from lactating animals revealed an elevation of genes known to be utilized by osteoclasts to remove bone including Tartrate Resistant Acid Phosphatase, TRAP, and cathepsin K that returned to virgin levels upon weaning. Infusion of Parathyroid Hormone Related Peptide, PTHrP, known to be elevated during lactation, induced TRAP activity and cathepsin K expression in osteocytes concurrent with osteocytic remodeling. Conversely, animals lacking the Parathyroid Hormone Type 1 receptor, PTHR1, in osteocytes failed to express TRAP or cathepsin K or to remodel their osteocyte perilacunar matrix during lactation. These studies show that osteocytes remove mineralized matrix through molecular mechanisms similar to those utilized by osteoclasts.

We have demonstrated that cytokine thymic stromal lymphopoietin (TSLP), whose expression is rapidly induced upon keratinocyteselective ablation of retinoid X receptors (RXRs) -␣ and -␤ in the mouse (RXR␣␤ ep؊/؊ mice), plays a key role in initiating a skin and systemic atopic dermatitis-like phenotype. We show here that topical application of the physiologically active ligand [1␣,25-(OH) 2D3; calcitriol] of the vitamin D receptor, or of its low-calcemic analog MC903 (calcipotriol; Dovonex), induces TSLP expression in epidermal keratinocytes, which results in an atopic dermatitis-like syndrome mimicking that seen in RXR␣␤ ep؊/؊ mutants and transgenic mice overexpressing TSLP in keratinocytes. Furthermore, topical application of retinoic acid receptor RAR␥-selective agonist BMS961 also induces TSLP expression either on its own or synergistically with 1␣,25-(OH)2D3. Our data demonstrate that RXR͞ vitamin D receptor and RXR͞retinoic acid receptor-␥ heterodimers and their ligands cell-autonomously control the expression of TSLP in epidermal keratinocytes of the mouse. We propose molecular mechanisms through which vitamin D3 and retinoic acid signalings could be involved in the pathogenesis of atopic diseases.retinoic acid ͉ vitamin D receptor ͉ retinoid X receptor ͉ retinoic acid receptor ͉ skin N uclear receptors (NRs) belong to a superfamily of liganddependent transcriptional regulators (1, 2). Within this superfamily, retinoid X receptors (RXRs) -␣, -␤, and -␥ play a key role through heterodimerization with some 15 NR partners, e.g., retinoic acid receptors (RARs), vitamin D receptor (VDR), peroxisome proliferator-activated receptors, and liver X receptors (1, 2). We reported (3) that selective ablation of RXR␣ and RXR␤ in adult mouse epidermal keratinocytes (RXR␣␤ epϪ/Ϫ mice) triggers a skin and systemic syndrome similar to human atopic dermatitis (AD), a chronic skin inflammatory disease with a strong genetic component that affects children (10-20%) and adults (1-3%) (4). These mice exhibit the major features of the human AD syndrome that include (i) skin eczematous-like lesions with xerosis and pruritus, associated with a skin inflammatory infiltrate mainly composed of CD4 ϩ T helper (Th) type 2 cells, dendritic cells, eosinophils, and mast cells and (ii) systemic abnormalities, including elevated serum IgE and IgG levels and blood and tissue eosinophilia.We found that expression of the cytokine thymic stromal lymphopoietin (TSLP), known to be produced in epidermal keratinocytes of AD patients (5), is rapidly induced in keratinocytes of RXR␣␤ epϪ/Ϫ mice. Furthermore, we showed that K14-TSLP transgenic mice overexpressing TSLP in keratinocytes exhibit an ADlike phenotype similar to that of RXR␣␤ epϪ/Ϫ mice (3), demonstrating that TSLP can act as an initiating cytokine at the top of a chain of immunological events that lead to an AD-like phenotype, in keeping with other recent studies on mouse models of human allergic inflammatory diseases (asthma and AD) (6-9).We suggested that up-regulation of keratinocytic TSLP...

Environmental contaminants affect a wide variety of biological events in many species. Dioxins are typical environmental contaminants that exert adverse oestrogen-related effects. Although their anti-oestrogenic actions are well described, dioxins can also induce endometriosis and oestrogen-dependent tumours, implying possible oestrogenic effects. However, the molecular mechanism underlying oestrogen-related actions of dioxins remains largely unknown. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix-loop-helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Here we show that the agonist-activated AhR/Arnt heterodimer directly associates with oestrogen receptors ER-alpha and ER-beta. This association results in the recruitment of unliganded ER and the co-activator p300 to oestrogen-responsive gene promoters, leading to activation of transcription and oestrogenic effects. The function of liganded ER is attenuated. Oestrogenic actions of AhR agonists were detected in wild-type ovariectomized mouse uteri, but were absent in AhR-/- or ER-alpha-/- ovariectomized mice. Our findings suggest a novel mechanism by which ER-mediated oestrogen signalling is modulated by a co-regulatory-like function of activated AhR/Arnt, giving rise to adverse oestrogen-related actions of dioxin-type environmental contaminants.