Acute bacterial infections and HIV disease

Hox homeobox genes play a crucial role in specifying the embryonic body pattern. However, a role for Hox genes in T-cell development has not been explored. The Hoxa-9 gene is expressed in normal adult and fetal thymuses. Fetal thymuses of mice homozygous for an interruption of the Hoxa-9 gene are one eighth normal size and have a 25-fold decrease in the number of primitive thymocytes expressing the interleukin-2 receptor (IL-2R, CD25). Progression to the double positive (CD4+CD8+) stage is dramatically retarded in fetal thymic organ cultures. This aberrant development is associated with decreased amounts of intracellular CD3 and T-cell receptor beta (TCRbeta) and reduced surface expression of IL-7R and E-cadherin. Mutant thymocytes show a significant increase in apoptotic cell death and premature downregulation of bcl-2 expression. A similar phenotype is seen in primitive thymocytes from adult Hoxa-9-/- mice and from mice transplanted with Hoxa-9-/- marrow. Hoxa-9 appears to play a previously unsuspected role in T-cell ontogeny by modulating cell survival of early thymocytes and by regulating their subsequent differentiation.     

Immunocytochemical localization of transforming growth factor alpha, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.     

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.     

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.     

Inhibition of DLX-7 homeobox gene causes decreased expression of GATA-1 and c-myc genes and apoptosis

The DLX gene family is a family of divergent homeobox genes which are related to the Drosophila distal-less (Dll) gene and has been reported to be expressed primarily in the forebrain and craniofacial structures. We have previously identified a new member of this family, DLX-7. We now report that this gene is expressed in normal hematopoietic cells and leukemia cell lines with erythroid characteristics. We used an antisense oligonucleotide targeted against the translation start site of DLX-7 mRNA to inhibit its expression in a human erythroleukemia cell line K562, which expresses DLX-7 at a high level. The antisense oligonucleotide efficiently reduced the DLX-7 mRNA, while control oligonucleotides, including a mutant oligonucleotide identical to the antisense sequence except for four nucleotide mismatches, had no effect on DLX-7 mRNA level. Inhibition of DLX-7 expression decreased the plating efficiency by approximately 70% compared with control. The antisense treatment caused apoptosis, as shown by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) method. Down-regulation of DLX-7 expression by antisense treatment was associated with a reduction in GATA-1 and c-myc mRNA levels. Thus, we conclude that DLX-7 is expressed in hematopoietic cells and that the inhibition of its expression results in the decreased levels of GATA-1 and c-myc genes, with an accompanying induction of apoptosis.     

Expression of a soluble form of LFA-1 and demonstration of its binding activity with ICAM-1

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.     

Lactation-dependent expression of an mRNA splice variant with an exon for a multiply O-glycosylated domain of mouse milk fat globule glycoprotein MFG-E8

Expression of mRNA encoding MFG-E8, a milk fat-associated glycoprotein was investigated in mouse mammary gland. Two forms of mRNA, long and short variants, were shown to be expressed in the mammary tissue by RT-PCR analysis. Sequence analyses of these two variants and an isolated MFG-E8 gene segment indicated that the long and short mRNA variants resulted from an alternative splicing of a single pre-mRNA through in-flame inclusion and skipping of one exon, which encodes a proline/threonine (Pro/Thr)-rich domain. The long variant was expressed predominantly in mammary gland and the expression level was remarkably increased at late gestation and kept high during lactation. On the contrary, the short variant was detected ubiquitously in various tissues and its expression in the mammary gland was rather decreased in a lactation dependent manner. Expression of the long variant was also detected in a mouse mammary epithelial cell line, COMMA-1D, and enhanced by incubation with lactogenic hormones. Glycosylation inhibition analyses using tunicamycin and alpha-benzyl-GalNAc were conducted with COS7 cells transfected with plasmids expressing each mRNA variant, demonstrating that a fully glycosylated product of the long mRNA variant was not only N-glycosylated but also multiply O-glycosylated, whereas a product of the short one had only N-glycan(s). These results suggest that the alternative splicing plays a critical role for the mammary-specific and lactation-dependent expression of the MFG-E8 isoform and that the multiply O-glycosylated Pro/Thr-rich domain of this isoform is functionally important for formation of milk fat globules in mammary epithelial cells.     

Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation

To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell-cell and cell-substratum interactions and in transduction process: indeed, E-cadherin, integrin-beta4 subunit, laminin-gamma2 chain, hemidesmosomal protein, APC, and alpha-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures. Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-alpha1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-alpha1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix-mediated intestinal cell differentiation.     

Chorionic gonadotropin inhibits rat mammary carcinogenesis through activation of programmed cell death

Human chorionic gonadotropin (hCG) inhibits the progression of 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary carcinomas. In order to determine whether this phenomenon was mediated by induction of programmed cell death or apoptosis, 45-day-old virgin Sprague-Dawley rats received 8 mg DMBA/100 g body weight; 20 days later they were injected daily with 100 IU hCG for 40 days (DMBA + hCG group). Age-matched untreated, hCG- and DMBA + saline treated rats were used as controls. Tissues were collected at the time of DMBA administration and at 5, 10, 20 and 40 days of hCG injection. RNA from mammary glands, adenocarcinomas and ovaries was probed for transforming growth factors (TGF) alpha and beta, and the apoptotic genes TRPM2, ICE, bcl2, bcl-XL, bcl-XS, p53 and c-myc. The mammary glands of hCG-treated animals with or without DMBA exhibited elevated expression of TRPM2, ICE, bcl-XS, c-myc and p53; and elevation in the apoptotic index. Mammary adenocarcinomas developed in those animals treated with hCG showed an elevation in the expression of p53, c-myc and ICE genes in comparison with the levels detected in the adenocarcinomas developed by the animals treated with DMBA alone. No significant alterations in the expression of any of the genes tested was observed in ovarian RNAs. These results led us to conclude that hCG induces programmed cell death in the mammary gland initiated in the carcinogenic process, that this process is p53 dependent, and is modulated by c-myc expression. Our data also indicate the possibility that a cell death program dependent on the bcl2 family exists, because of the potential involvement of p53, bcl-XS and Bax in apoptosis. This additional mechanism of tumor inhibition makes hCG treatment a useful approach for the prevention and therapy of breast cancer.     

decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary

The purpose of the present studies was to investigate the role of epidermal growth factor (EGF) in the acquisition of estrogen (E) and progestin (P) responsiveness in the mouse mammary gland in vivo. Using the Elvax 40P implant technique to introduce bioactive molecules directly into the mammary gland to produce a localized effect, we have made the novel observation that EGF implanted into glands of pubertal mice followed by E treatment resulted in the precocious acquisition of E-inducible progesterone receptors (PR). In sexually mature mice, EGF implants alone were able to increase PR. A neutralizing antibody specific for EGF blocked E-dependent stimulation of end-bud development and PR induction. Furthermore, the antiestrogen ICI 182,780 blocked the EGF-induced stimulation end-buds and PR induction, indicating that these EGF effects are mediated via estrogen receptors (ER). Immunohistochemical analysis showed that the endogenous EGF content of mammary glands of mature mice was higher than pubertal mice, that E implants caused a localized increase in mammary gland EGF content in both pubertal and mature mice, and that in mature mice E caused an increase in stromal cell EGF content. We have previously shown that the acquisition of E-inducible PR can be modulated by mammary stroma, and the present results indicate that mammary stroma could modulate hormonal responsiveness through control of local growth factor concentration. Taken together, these results provide evidence that E-dependent responses of mouse mammary gland in vivo, such as end-bud proliferation and PR regulation, may be mediated by EGF through an ER-dependent mechanism.     

Radiation-induced alteration of rat mesangial cell transforming growth factor-beta and expression of the genes associated with the extracellular matrix

We hypothesized that the altered mesangial cell phenotype observed in radiation nephropathy reflects, at least partly, radiation-induced changes in expression of the genes associated with transforming growth factor-beta (TGF-beta) and extracellular matrix (ECM). To test this hypothesis, rat mesangial cells were used between passages 7 and 11 after primary isolation from glomeruli. Cells were placed in serum-free medium 24 h prior to irradiation and irradiated with single doses of 5-20 Gy 137Cs gamma rays; control cells received sham irradiation. After irradiation, the cells were maintained in serum-free medium for up to 48 h postirradiation. Total RNA was isolated, and Northern analysis was performed using cDNA probes for TGF-beta 1, beta 2 and beta 3 and several ECM genes. Irradiation resulted in isoform-specific alterations in TGF-beta mRNA; TGF-beta 1 levels showed a dose-independent increase 24-48 h postirradiation; TGF-beta 3 mRNA levels showed a progressive dose-independent decrease over the same period, decreasing to levels approximately 25% of those seen in controls. These changes were associated with a concomitant increase in levels of mRNA expressed by genes for the components of the ECM; no changes were observed in TGF-beta 2, collagen I, collagen III or decorin. Thus radiation can alter mesangial cell TGF-beta and the expression of the genes involved in ECM, although the nature of this alteration varies for the TGF-beta isoforms and specific ECM genes.     

Heterochronic differences of Hoxa-11 expression in Xenopus fore- and hind limb development: evidence for lower limb identity of the anuran ankle bones

The wrist (carpus) and ankle (tarsus) of most tetrapods, as well as the wrist of anurans, contains relatively small nodular skeletal elements. The anuran tarsus, however, comprises a pair of long bones, the proximal tarsals tibiale and fibulare, which resemble the lower leg bones, tibia and fibula (zeugopodium). In this paper we investigate whether the proximal tarsals of Xenopus are of zeugopodial character identity, i.e. whether they develop under the influence of the same genes that pattern the lower limb. We compare Hoxa-11 expression in the forelimb bud with that in the hind limb bud by whole-mount in situ hybridization. Hoxa-11 has been implicated in the development of the lower limb. In Xenopus we note three differences between Hoxa-11 expression in fore- and hind limb buds: (1) Hoxa-11 expression is maintained until the hind limb bud reaches a larger size (2 mm) than that of the forelimb bud (1.5 mm); (2) Hoxa-11 expression is maintained over larger spatial domains than in the forelimb; and (3) Hoxa-11 expression has a pronounced posterior polarity in the hind limb, but not in the forelimb. Hind limb expression of Hoxa-11 can be understood as a heterochronic prolonging of the expression dynamic in the forelimb. Finally we found that the proximal tarsals start to develop within the expression domain of Hoxa-11, while in the forelimb the lower arm elements reach the distal expression limit of Hoxa-11. The gene expression data presented here support the notion of a zeugopodial identity of the proximal tarsal elements in Xenopus.     

A homeobox gene with potential developmental control function in the meristem of the conifer Picea abies

Many homeobox genes control essential developmental processes in animals and plants. In this report, we describe the first cDNA corresponding to a homeobox gene isolated from a gymnosperm, the HBK1 gene from the conifer Picea abies (L.) Karst (Norway spruce). The sequence shows distinct similarities specifically to the KNOX (knotted-like homeobox) class of homeobox genes known from different angiosperm plants. The deduced amino acid sequence of HBK1 is strikingly similar within the homeodomain (84% identical) to the maize gene Knotted1 (Kn1), which acts to regulate cell differentiation in the shoot meristem. This similarity suggested that the phylogenetic association of HBK1 with the KNOX genes might be coupled to a conservation of gene function. In support of this suggestion, we have found HBK1 to be expressed in the apical meristem in the central population of nondifferentiated stem cells, but not in organ primordia developing at the flanks of the meristem. This pattern of expression is similar to that of Kn1 in the maize meristem. We show further that HBK1, when expressed ectopically in transgenic Arabidopsis plants, causes aberrations in leaf development that are similar to the effects of ectopic expression of angiosperm KNOX genes on Arabidopsis development. Taken together, these data suggest that HBK1 has a role, similar to the KNOX genes in angiosperms, in the control of cellular differentiation in the apical meristem of spruce. The data also indicate that KNOX-gene regulation of vegetative development is an ancient feature of seed plants that was present in the last common ancestor of conifers and angiosperms.     

How many homeobox genes does it take to make a pituitary gland?

The secrets of anterior pituitary development and cell lineage determination have been revealed mostly by genetic analyses. The requirement for three homeobox genes, Lbx3, Lbx4 and Titf1, during early organogenesis was proven by gene targeting. Spontaneous mouse mutations revealed two additional homeobox genes, Pit1 and Prop1, that are critical for specialization and proliferation of subsets of the five differentiated cell types. Analysis of patients with pituitary insufficiency has demonstrated the importance of these two genes in human pituitary function. Recently, several other homeobox genes have been identified and implicated in pituitary organogenesis. Genetic manipulations of these genes will undoubtedly add to the emerging genetic hierarchy regulating the ontogeny of this major hormone-producing gland.     

Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development

Parathyroid hormone-related protein (PTHrP) was originally discovered as a tumor product that causes humoral hypercalcemia of malignancy. PTHrP is now known to be widely expressed in normal tissues and growing evidence suggests that it is an important developmental regulatory molecule. We had previously reported that overexpression of PTHrP in the mammary glands of transgenic mice impaired branching morphogenesis during sexual maturity and early pregnancy. We now demonstrate that PTHrP plays a critical role in the epithelial-mesenchymal communications that guide the initial round of branching morphogenesis that occurs during the embryonic development of the mammary gland. We have rescued the PTHrP-knockout mice from neonatal death by transgenic expression of PTHrP targeted to chondrocytes. These rescued mice are devoid of mammary epithelial ducts. We show that disruption of the PTHrP gene leads to a failure of the initial round of branching growth that is responsible for transforming the mammary bud into the rudimentary mammary duct system. In the absence of PTHrP, the mammary epithelial cells degenerate and disappear. The ability of PTHrP to support embryonic mammary development is a function of amino-terminal PTHrP, acting via the PTH/PTHrP receptor, for ablation of the PTH/PTHrP receptor gene recapitulates the phenotype of PTHrP gene ablation. We have localized PTHrP expression to the embryonic mammary epithelial cells and PTH/PTHrP receptor expression to the mammary mesenchyme using in situ hybridization histochemistry. Finally, we have rescued mammary gland development in PTHrP-null animals by transgenic expression of PTHrP in embryonic mammary epithelial cells. We conclude that PTHrP is a critical epithelial signal received by the mammary mesenchyme and involved in supporting the initiation of branching morphogenesis.