Characterization of rare mammary tumours appearing on the neck of RIII/Sa mice infected with mouse mammary tumour virus.

RIII/Sa and C3H mice harbour milk-borne mouse mammary tumour virus (MMTV) and develop mammary tumours at a high incidence. These mammary tumours usually arise ventrally and/or on the sides of the animals. In the present study, some mice of both strains were observed to have tumours in the dorsal neck area. Histological analysis of the tumours indicated their similarity to mammary tumours induced by MMTV oncogenesis. The neck tumours were found by thin-section electron microscopy to contain both type A and type B particles that are hallmarks of MMTV infection. In addition, the neck tumour DNA possessed insertion mutations of Wnt-1 and Fgf-3 proto-oncogenes, the activation of which play important roles in the development of mouse mammary tumours. These neck tumours appear to be mammary tumours that arise in the context of in-situ mammary tissue, similar to rare 'ectopic' human breast cancers that arise in the axillary region and other sites remote from the breast.

Tbx2 represses expression of Connexin43 in osteoblastic-like cells.

Tbx2 belongs to a family of developmental transcription regulatory factors. We evaluated whether the gap junction protein Connexin43 (Cx43), an important regulator of osteoblast function and bone development, may be a downstream target gene regulated by Tbx2. The Cx43 promoter contains direct repeats of the consensus T-box binding motif, TCACAC, and moreover, Tbx2 and Cx43 show overlapping expression domains in precursors to bone and in osteoblasts. In vitro analysis showed that the Cx43 promoter contains two Tbx2 binding sites, and this binding was dependent on the TCACAC consensus sequence. Transient transfection analysis with a Cx43 promoter-driven lacZ reporter construct revealed negative regulation mediated by these two Tbx2 binding sites in osteoblast-like cells. Thus, downregulation of Tbx2 led to de-repression of wild-type Cx43 promoter activity, whereas a promoter construct with mutated binding sites showed no de-repression. In stably transfected osteosarcoma cells in which expression of the endogenous Tbx2 gene was downregulated with a Tbx2 antisense construct, a marked de-repression of the endogenous Cx43 gene was observed. This was accompanied by a marked increase in the abundance of Cx43 gap junctions and increased functional gap junction-mediated cell-cell communication. Analysis of lacZ expression in transgenic mice containing the mutated Cx43 promoter-driven lacZ construct further suggested de-repression of the Cx43 promoter in limb buds, a region destined to give rise to long bones of the limbs. Taken together, these findings indicate that the promoter of Cx43 is repressible by Tbx2, both in cultured osteoblast-like cells in vitro and likely in the developing embryo.

Microarray analysis of Tbx2-directed gene expression: a possible role in osteogenesis.

Tbx2 is a member of the developmentally important transcriptional regulatory T-box gene family, whose target genes have not been well characterized. In an attempt to identify genes that may be regulated by Tbx2, mouse cDNA microarrays were used to analyze differential gene expression profiles, comparing stably transfected NIH3T3 cells overexpressing Tbx2 and vector-transfected controls. Among 8734 genes, 107 genes were up-regulated by 2-fold or greater, and 66 genes were down-regulated by 2-fold or greater. Caveolin, pleiotrophin (osf-1), osteoblast-specific factor-2 (osf-2) and collagen type I alpha were among the genes upregulated in the Tbx2-overexpressing cells, whereas cadherin 3, tenascin C, and insulin-like growth factor binding protein 10/CYR61 (IBP10) were among the genes downregulated. Northern blot analysis confirmed the correlation of expression of several genes, including IBP10 and osf-2, in fibroblast NIH3T3 and rat osteosarcoma ROS17/2.8 cells differentially expressing Tbx2. In ROS17/2.8 cells transfected with antisense Tbx2, osf-2 was downregulated, whereas transfection of sense Tbx2 upregulated this gene. Interestingly, the expression of pleiotrophin (osf-1) and collagen I alpha with Tbx2 transfection showed an inverse regulatory correlation between NIH3T3 and ROS17/2.8 cells. Thus, Tbx2 can act as both a repressor and activator, and the cellular context can influence the effect on gene expression. Although the data do not address whether Tbx2 directly mediates the transcriptional effect, a number of candidate genes possess putative T-box gene regulatory elements. The results support the hypothesis that Tbx2 may be an important modulator of bone development. Further functional cluster analysis indicates that Tbx2 might also be involved in the regulation of cell cycle and cell adhesion.

Glucose-dependent insulinotropic peptide is an integrative hormone with osteotropic effects.

Glucose-dependent insulinotropic peptide (GIP) is a gut-derived hormone known to be important in modulating glucose-induced insulin secretion. In addition, GIP receptors are widely distributed and may have effects on multiple other tissues: fat cells, adrenal glands, endothelium and brain. We have demonstrated recently that GIP also has anabolic effects on bone-derived cells. We now demonstrate that GIP administration prevents the bone loss associated with ovariectomy. We propose that GIP plays a unique role in signaling the bone about nutrient availability, indicating the importance of the gut hormones in directing absorbed nutrients to the bone, and suggesting the concept of an 'entero-osseous axis'. Thus, GIP plays an integrative role helping coordinate efficient and targeted nutrient absorption and distribution.

1,25-Dihydroxyvitamin D(3), phospholipase D and protein kinase C in keratinocyte differentiation.

1,25-Dihydroxyvitamin D(3), thought to be a physiological regulator of epidermal keratinocyte growth and differentiation, also elicits the complete differentiative program in vitro, with expression of various genes/proteins characteristic of both early and late differentiation. 1,25-Dihydroxyvitamin D(3) functions by interacting with an intracellular receptor that binds to DNA at vitamin D response elements (VDRE) thereby affecting transcription. 1,25-Dihydroxyvitamin D(3) has been demonstrated to alter the expression of several enzymes involved in signal transduction, and presumably this is the mechanism through which the hormone regulates differentiation. It has recently been shown that 1,25-dihydroxyvitamin D(3) specifically increases the expression/activity of phospholipase D-1, an enzyme that hydrolyzes phospholipids to generate lipid messengers, such as diacylglycerol (DAG). DAG, in turn, is known to activate several members of the protein kinase C (PKC) family. It has been proposed that this signaling pathway mediates late differentiation events in epidermal keratinocytes. In this article the data supporting a role for PKC and phospholipase D in keratinocyte differentiation, as well as in the pathogenesis of skin diseases, are reviewed and a model is proposed for the signaling pathways that regulate this process upon exposure to 1,25-dihydroxyvitamin D(3).

Functional analysis of glucose-dependent insulinotropic polypeptide fusion proteins.

To generate functional fluorescently tagged glucose-dependent insulinotropic polypeptide (GIP), a series of GIP expression constructs were devised. These included G1 (complete preprohormone), G2 (lacking the C-terminal extension), G3 (lacking both N- and C-terminal extensions), G4 (G2 fused to green fluorescent protein, GFP), and G5 (G3 fused to GFP). Expression of G5 in bacteria generated immunopositive GIP together with GFP fluorescence, while G4 generated only fluorescence without immunoreactivity. Transfection of NIH3T3 cells with cDNAs of G1, G3, G5, but not G2, G4, and EGFP, resulted in immunologically detectable GIP formation, although fluorescence could be detected in the latter two. GIP as well as GIP-GFP secreted by NIH3T3 cells significantly stimulated intracellular cAMP accumulation and Ca(2+) mobilization in SaOS2 cells. The GIP receptor antagonist GIP(7-30) abolished these responses. These results suggest that a GIP-GFP fusion protein seven times larger than the native peptide retains function and may be used as an in vivo probe to detect GIP receptor distribution and to explore GIP's biological roles.

Glucose-dependent insulinotropic peptide signaling pathways in endothelial cells.

Glucose-dependent insulinotropic peptide (GIP) potentiates glucose-induced insulin secretion. In addition, GIP has vasoconstrictive or vasodilatory properties depending on the vascular bed affected. In order to assess whether this effect could be related to differences in GIP receptor expression, several different endothelial cell types were examined for GIP receptor expression. GIP receptor splice variants were detected and varied depending on the endothelial cell type. Furthermore, stimulation of these cells with GIP led to cell type dependent differences in activation of the calcium and cAMP signaling pathways. To our knowledge this is the first physiological characterization of receptors for GIP in endothelial cells.

Functional parathyroid hormone receptors are present in an umbilical vein endothelial cell line.

Acute parathyroid hormone exposure induces vascular smooth muscle relaxation. In contrast, continuous infusion of parathyroid hormone leads to vasoconstriction and an elevation of blood pressure. Despite the known effects of parathyroid hormone on vascular smooth muscle, possible direct effects on the vascular endothelium have not previously been investigated. Using a human umbilical vein endothelial cell line, we found that parathyroid hormone increased both intracellular calcium and cellular cAMP content in these endothelial cells. Furthermore, exposure of these cells to increasing concentrations of parathyroid hormone stimulated both [(3)H]thymidine incorporation and endothelin-1 secretion. Parathyroid hormone/parathyroid hormone-related peptide receptor mRNA could be detected at low levels in these cells. In summary, these data demonstrate that endothelium-derived cells contain functional parathyroid hormone receptors. The potential physiological role of these receptors remains to be determined.

Osteoblast-derived cells express functional glucose-dependent insulinotropic peptide receptors.

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid peptide synthesized and secreted from endocrine cells in the small intestine. The role of GIP in coupling nutrient intake and insulin secretion, the incretin effect, is well known. We report that GIP receptor messenger RNA and protein are present in normal bone and osteoblast-like cell lines, and that high affinity receptors for GIP can be demonstrated by [125I]GIP binding studies. When applied to osteoblast-like cells (SaOS2), GIP stimulated increases in cellular cAMP content and intracellular calcium, with both responses being dose dependent. Moreover, administration of GIP results in elevated expression of collagen type I messenger RNA as well as an increase in alkaline phosphatase activity. Both of these effects reflect anabolic actions of presumptive osteoblasts. These results provide the first evidence that GIP receptors are present in bone and osteoblast-like cells and that GIP modulates the function of these cells.

Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras.

Monitoring the migrations of cells during embryonic development requires a system in which cells can be identified in situ during locomotion. One promising system involves the generation of chimeras by transplanting mouse cells into chick embryos in ovo to exploit the wealth of mouse genetic variants. The success of this technique relies on the ability to detect individual mouse cells in a chick environment with high specificity. The murine B2 family of short interspersed elements is present in the mouse genome at copy numbers in excess of 10(5), whereas this sequence is absent in the chick genome based on hybridization techniques. This differential of five orders of magnitude produces signals in mouse cells that are easily identified, even in an environment that is predominantly chick. Thus, the B2 repeat probe is highly effective for the purpose of identifying mouse cells in mouse-chick chimeras.

1,25-dihydroxyvitamin D3 induces phospholipase D-1 expression in primary mouse epidermal keratinocytes.

The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) elicits the programmed pattern of differentiation in epidermal keratinocytes. Based on data indicating a potential role of phospholipase D (PLD) in mediating keratinocyte differentiation, we investigated the effect of 1,25(OH)2D3 on PLD expression. A 24-h exposure to 1, 25(OH)2D3 stimulated PLD-1, but not PLD-2, mRNA expression. This 1, 25(OH)2D3-enhanced expression was accompanied by increased total PLD and PLD-1 activity. Time course studies indicated that 1,25(OH)2D3 induced PLD-1 expression by 8 h, with a maximal increase at 20-24 h. Exposure to 1,25(OH)2D3 inhibited proliferation over the same time period with similar kinetics. Expression of the early (spinous) differentiation marker keratin 1 decreased in response to 1, 25(OH)2D3 over 12-24 h. Treatment with 1,25(OH)2D3 enhanced the activity of transglutaminase, a late (granular) differentiation marker, by 12 h with a maximal increase after 24 h. In situ hybridization studies demonstrated that the highest levels of PLD-1 expression are in the more differentiated (spinous and granular) layers of the epidermis, with little expression in basal keratinocytes. Our results suggest a role for PLD expression/activity during keratinocyte differentiation.

Cloning and expression analysis of murine phospholipase D1.

Activation of phosphatidylcholine-specific phospholipase D(PLD) occurs as part of the complex signal-transduction cascade initiated by agonist stimulation of tyrosine kinase and G-protein-coupled receptors. A variety of mammalian PLD activities have been described, and cDNAs for two PLDs recently reported (human PLD1 and murine PLD2). We describe here the cloning and chromosomal localization of murine PLD1. Northern-blot hybridization and RNase protection analyses were used to examine the expression of murine PLD1 and PLD2 ina variety of cell lines and tissues. PLD1 and PLD2 were expressed in all RNA samples examined, although the absolute expression of each isoform varied, as well as the ratio of PLD1 to PLD2. Moreover, in situ hybridization of adult brain and murine embryo sections revealed high levels of expression of individual PLDs in some cell types and no detectable expression in others. Thus the two PLDs probably carry out distinct roles in restricted subsets of cells rather than ubiquitous roles in all cells.

Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development.

A novel family of genes, characterized by the presence of a region of homology to the DNA-binding domain of the Brachyury (T) locus product, has recently been identified. The region of homology has been named the T-box, and the new mouse genes that contain the T-box domain have been named T-box 1-6 (Tbx1 through Tbx6). As the basis for further study of the function and evolution of these genes, we have examined the expression of 5 of these genes, Tbx1-Tbx5, across a wide range of embryonic stages from blastocyst through gastrulation and early organogenesis by in situ hybridization of wholemounts and tissue sections. Tbx3 is expressed earliest, in the inner cell mass of the blastocyst. Four of the genes are expressed in different components of the mesoderm or mesoderm/endoderm during gastrulation (Tbx1 and Tbx3-5). All of these genes have highly specific patterns of expression during later embryogenesis, notably in areas undergoing inductive tissue interactions. In several cases there is complementary expression of different genes in 2 interacting tissues, as in the lung epithelium (Tbx1) and lung mesenchyme (Tbx2-5), and in mammary buds (Tbx3) and mammary stroma (Tbx2). Tbx1 shows very little overlap in the sites of expression with the other 4 genes, in contrast to a striking similarity in expression between members of the 2 cognate gene sets, Tbx2/Tbx3 and Tbx4/Tbx5. This is a clear reflection of the evolutionary relationship between the 5 genes since the divergence of Tbx1 occurred long before the relatively recent divergence of Tbx2 and 3 and Tbx4 and 5 from common ancestral genes. These studies are a good indication that the T-box family of genes has important roles in inductive interactions in many stages of mammalian embryogenesis.

Conservation of the T-box gene family from Mus musculus to Caenorhabditis elegans.

Recently, a novel family of genes with a region of homology to the mouse T locus, which is known to play a crucial, and conserved, role in vertebrate development, has been discovered. The region of homology has been named the T-box. The T-box domain of the prototypical T locus product is associated with sequence-specific DNA binding activity. In this report, we have characterized four members of the T-box gene family from the nematode Caenorhabditis elegans. All lie in close proximity to each other in the middle of chromosome III. Homology analysis among all completely sequenced T-box products indicates a larger size for the conserved T-box domain (166 to 203 residues) than previously reported. Phylogenetic analysis suggests that one C. elegans T-box gene may be a direct ortholog of the mouse Tbx2 and Drosophila omb genes. The accumulated data demonstrate the ancient nature of the T-box gene family and suggest the existence of at least three separate T-box-containing genes in a common early metazoan ancestor to nematodes and vertebrates.

Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells.

We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homology or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected.

An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus.

The T locus encodes a product with DNA binding activity that is likely to play a role in the development of all vertebrate organisms. We have identified and characterized a novel family of mouse genes that share a protein motif, the T-box, with the prototypical T locus. The T-box domain of the T locus co-localizes with its DNA binding activity. Each T-box gene is expressed in a unique temporal and spatial pattern during embryogenesis. Phylogenetic analysis suggests that at least three T-box genes were present in the common ancestor to vertebrates and invertebrates. Thus, members of the T-box family could have played a role in the evolution of all metazoan organisms.

Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.

We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.

Direct-repeat analysis of chromatid interactions during intrachromosomal recombination in mouse cells.

Homologous intrachromosomal recombination between linked genes can involve interactions that are either intramolecular (intrachromatid) or intermolecular (sister chromatid). To assess the relative proportions of chromatid interactions, we report studies of intrachromosomal recombination in mouse L cells containing herpes simplex virus thymidine kinase genes in two alternative configurations of direct repeats. By comparing products of reciprocal exchanges between these two configurations, we conclude that the majority of interactions that give rise to crossover products involve unequally paired sister chromatids after DNA replication. Analyses of an additional class of crossover products that involve discontinuous associated gene conversion suggest that these recombination events involve a heteroduplex DNA intermediate.

Conservative intrachromosomal recombination between inverted repeats in mouse cells: association between reciprocal exchange and gene conversion.

Recombination in mammalian cells is thought to involve both reciprocal and nonreciprocal modes of exchange, although rigorous proof is lacking due to the inability to recover all products of an exchange. To investigate further the relationship between these modes of exchange, we have analyzed intrachromosomal recombination between duplicated herpes simplex virus thymidine kinase (HSV tk) mutant alleles arranged as inverted repeats in cultured mouse L cells. In crosses between inverted repeats, a single intrachromatid reciprocal exchange leads to inversion of the sequence between the crossover sites and recovery of both genes involved in the event. The majority of recombinant products do not display such inversion and are thus consistent with a nonreciprocal mode of recombination (gene conversion). The remaining products display the sequence inversion predicted for intrachromatid reciprocal exchange. In light of the fact that intrachromatid exchanges occur, the rarity of intrachromatid double reciprocal exchanges strengthens the interpretation that the majority of events in this and previous investigations involve gene conversion. Furthermore, in accord with prediction, one-third of the reciprocal recombinants (inversions) display associated gene conversion. This association suggests that reciprocal and nonreciprocal modes of exchange are mechanistically related in mammalian cells. Finally, the occurrence of inversion recombinants suggests that intrachromosomal recombination can be a conservative (nondestructive) process.