Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin.

The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2-/- MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2-/- MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.

Stem cells in the embryonic kidney.

The mammalian kidney, the metanephros, is formed by a reciprocally inductive interaction between two precursor tissues, the metanephric mesenchyme and the ureteric bud. The ureteric bud induces the metanephric mesenchyme to differentiate into the epithelia of glomeruli and renal tubules. Multipotent renal progenitors that form colonies upon Wnt4 stimulation and strongly express Sall1 exist in the metanephric mesenchyme; these cells can partially reconstitute a three-dimensional structure in an organ culture setting. Six2 maintains this mesenchymal progenitor population by opposing Wnt4-mediated epithelialization. Upon epithelial tube formation, Notch2 is required for the differentiation of proximal nephron structures (podocyte and proximal tubules). In addition, the induction methods of the intermediate mesoderm, the precursor of the metanephric mesenchyme, begin to be elucidated. If derivation of metanephric mesenchyme becomes possible, we will be closer to the generation and manipulation of multiple cell lineages in the kidney.

Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development.

SALL1 is a mammalian homolog of the Drosophila region-specific homeotic gene spalt (sal); heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We have isolated a mouse homolog of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1-deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development.

A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages.

Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons.

Cloning and expression pattern of a Xenopus pronephros-specific gene, XSMP-30.

The first step in kidney development is the formation of the pronephros which is derived from mesoderm. Xenopus is an appropriate model to study this process since the pronephros can be efficiently induced in animal cap explants by treatment with activin and retinoic acid (RA). Using this in vitro system, we isolated a Xenopus homologue of SMP-30 (Senescence marker protein-30), which is a Ca(2+)-binding protein that is highly conserved in vertebrates. This gene, termed XSMP-30, was found to be selectively expressed in pronephric tubules from the late tadpole stage, by whole mount in situ hybridization. Furthermore XSMP-30 was expressed in animal caps treated with both activin and RA, a condition in which the pronephros is formed in vitro. These data indicate that XSMP-30 is a specific marker for the pronephros.

Molecular cloning of a novel Xenopus spalt gene (Xsal-3).

The sal (spalt) gene family is characterized by unique double zinc finger motifs and is conserved among various species from Drosophila to humans. Here we report a new Xenopus member of this family, Xsal-3. It is 38% homologous at the amino acid level to the previously reported Xenopus homologue of the spalt gene, Xsal-1. Alternatively spliced Xsal-3 transcripts give rise to RNAs coding either two or three double zinc fingers, and the longer form is expressed maternally. Xsal-3 is expressed in the neural tube, the mandibular, hyoid, and branchial arch, and the pronephric duct, which is different from the expression pattern of Xsal-1. These findings suggest that Xsal-3 may have distinct roles in early Xenopus development.

Human osteoclast-like cells are formed from peripheral blood mononuclear cells in a coculture with SaOS-2 cells transfected with the parathyroid hormone (PTH)/PTH-related protein receptor gene.

Subclones of the human osteosarcoma cell line SaOS-2 were established by transfecting with an expression vector containing the human PTH/PTH-related protein (PTHrP) receptor, and their abilities to support osteoclast-like multinucleated cell (OCL) formation were examined in coculture with mouse or human hemopoietic cells. Of four subclones examined, SaOS-2/4 and SaOS-4/3 bound high levels of [125I]-PTH and produced a significant amount of cAMP in response to PTH. OCLs were formed in response to PTH in the cocultures of mouse bone marrow cells with either SaOS-2/4 cells or SaOS-4/3 cells. Human OCLs were also formed in response to PTH in the coculture of SaOS-4/3 cells and human peripheral blood mononuclear cells. Adding dexamethasone together with PTH greatly enhanced PTH-induced human OCL formation. Like mouse OCLs, human OCLs formed in response to PTH were tartrate-resistant acid phosphatase positive, expressed abundant calcitonin receptors and vitronectin receptors, and formed resorption pits on dentine slices. Other osteotropic factors such as 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, and interleukin 6 plus soluble interleukin 6 receptors failed to induce mouse and human OCLs in cocultures with SaOS-4/3 cells. Both mouse and human OCL formation supported by SaOS-4/3 cells were inhibited by either adding an antibody against macrophage-colony stimulating factor or adding granulocyte/macrophage-colony stimulating factor. Thus, it is likely that human and mouse OCL formation supported by SaOS-4/3 cells are similarly regulated. These results indicate that the target cells of PTH for inducing osteoclast formation are osteoblast/stromal cells but not osteoclast progenitor cells in the coculture. This coculture model will be useful for investigating the abnormalities ofosteoclast differentiation and function in human metabolic bone diseases.

Activation of Stat3 by cytokine receptor gp130 ventralizes Xenopus embryos independent of BMP-4.

Stat3 is one of the main signaling components of cytokine receptors, including gp130. Here we show that activation of cytokine receptor gp130 resulted in a dramatic ventralization of Xenopus embryos and that the ventralization correlated well with Stat3 activation potential of the receptor. This finding led to identification of Xenopus Stat3 (Xstat3), which showed a 95% homology to its murine and human counterparts, at the amino acid level, and was expressed from the one-cell stage throughout development. The mechanism of gp130/XStat3-mediated ventralization proved to be independent of BMP-4. gp130/Xstat3 stimulation inhibited Smad2-induced ectopic axis formation in embryos and Smad2-dependent luciferase activity. A dominant-negative Stat3, in contrast, dorsalized Xenopus embryos, resulting in ectopic axis formation. We propose that Stat3-mediated signaling has the capacity to modify dorsoventral patterning in the early development of Xenopus.

The physiologic role of interleukin-3, interleukin-5, granulocyte-macrophage colony-stimulating factor, and the beta c receptor system.

Gene knockout techniques in the mouse have provided a unique opportunity to examine physiologic gene function. The phenotypes of mutant mice have resulted in a number of predicted as well as totally unexpected results, the latter perhaps providing the most valuable insight into otherwise unexplored biology. An excellent example to illustrate these points are gene knockout studies of interleukin (IL)-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), and the receptor system that cells use to bind these ligands. The role of IL-5 appears to be largely restricted to eosinophil biology, fulfilling expectations. However, the vivo function of GM-CSF, IL-3, and their receptor interaction has been more surprising. Neither cytokine nor their combination play an essential or even detectable role in primary bone marrow hematopoiesis, Instead, both IL-3 and GM-CSF play more specialized roles on certain cell subsets, basophil and mast cells, and alveolar macrophages, respectively. This review discusses recent findings with these mice, and based on these results, suggests a more appropriate view of the role of these cytokines and receptors in the hierarchy of molecular events leading to hematopoiesis.

Human pulmonary alveolar proteinosis associated with a defect in GM-CSF/IL-3/IL-5 receptor common beta chain expression.

Pulmonary alveolar proteinosis (PAP) is a heterogeneous disorder of genetic or acquired etiologies. In some cases congenital PAP is associated with hereditary surfactant protein (SP)-B deficiency. To date, the molecular defect in the majority of patients with PAP has not been identified. In mice, PAP has been generated by targeted deletion of the genes for either the GM-CSF/IL-3/IL-5 receptor common beta chain (beta c) or GM-CSF. Here, we describe an expression defect of beta c in three of seven pediatric patients with PAP and in one patient with severe lung disease suspected to be PAP. The patients failed to express normal levels of beta c as shown by flow cytometry. Strikingly reduced or absent function of beta c was demonstrated by ligand binding studies and progenitor clonogenic assays. Analysis of beta c DNA revealed a point mutation from proline to threonine at codon 602 in one patient. Our findings provide evidence that a defect in the expression of a hematopoietic cytokine receptor is associated with human PAP.

Xenopus FK 506-binding protein homolog induces a secondary axis in frog embryos, which is inhibited by coexisting BMP 4 signaling.

FK 506-binding protein (FKBP) is an immunosuppressant mediator in mammals, but its endogenous physiological function has yet to be determined. Here we report a Xenopus homolog of FKBP, which is expressed at early stages of development. Injection of synthesized Xenopus FKBP mRNA, as well as murine constitutively active calcineurin, induced a secondary axis in Xenopus embryos, while an FKBP mutant which does not bind to calcineurin did not. This secondary-axis-inducing effect was inhibited when FKBP was coinjected with Xmad 1 or XBMP 4 mRNA. These results suggest that FKBP modifies BMP 4 signalling by recruiting calcineurin and may have an important role in axis formation during Xenopus development.

The cytokine stew and innate resistance to L. monocytogenes.

The Listeria monocytogenes (L. monocytogenes) infection model has been a useful system to evaluate the cellular interactions leading to host immunity. The initiation of the innate immune response in naive animals and subsequent progression to acquired immunity represent an integrated system with numerous layers of complexity. Coincident with experimental infection is the induction of cytokines. Cytokines, which are soluble mediators of cell growth, maintenance and function, from a network of pleiotropic stimuli that serve as one of the main driving forces for the progressive development of cellular responses. A variety of in vivo approaches, such as injection of the recombinant cytokines themselves or antibodies to neutralize their activity, have been used to define these stimuli. Perhaps one of the most useful tools is that of germline-manipulated animals. One of the many cytokines implicated in resistance to L. monocytogenes infection is interleukin (IL)-6, a molecule associated with diverse infectious and pathophysiological disease states. This review concentrates on various cytokines (IL-1, TNF alpha, IFN-gamma, IL-12, IL-10 and the colony-stimulating factors (CSF)) thought to play a role during the innate host response to L. monocytogenes infection, with a special emphasis on studies using IL-6-deficient mice. Additionally, we show unpublished data obtained when the concepts learned from L. monocytogenes infection in IL-6-deficient mice were applied to other infection models.

Persistence of pulmonary pathology and abnormal lung function in IL-3/GM-CSF/IL-5 beta c receptor-deficient mice despite correction of alveolar proteinosis after BMT.

Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor (beta cR KO) develop lung disease similar to that seen in human pulmonary alveolar proteinosis (PAP) which includes lymphocytic infiltration around airways and vessels and the progressive accumulation of surfactant and macrophages within the alveolar space. We investigated bone marrow transplantation (BMT) as a curative treatment of PAP in beta cR KO mice by semiquantitative histologic analysis and evaluation of pulmonary function. BMT from wild-type (WT) donors into lethally irradiated beta cR KO recipients (WT --> KO) led to the complete resolution of alveolar protein accumulation and to normalization of BAL fluid cellularity and macrophage morphology. However, detailed microscopic analysis of lung tissue revealed the persistence of significant cellular infiltrates in WT --> KO recipients which were equivalent to those seen in KO --> KO animals. Evaluation of pulmonary function demonstrated that only dynamic compliance (Cdyn) and not airway conductance (G[L]) was significantly improved in the WT --> KO group compared to KO --> KO animals and that both of these measurements remained significantly abnormal when compared to WT --> WT controls. We conclude, that although BMT for PAP reverses alveolar macrophage and protein accumulation, it does not decrease the interstitial inflammatory component of this disease. The importance of this residual pathology is demonstrated by the incomplete correction of alveolar function (Cdyn) and lack of improvement in increased airway resistance (G[L]). These findings may have important implications with regard to the extent that BMT can be considered a potential curative procedure for this clinical disorder.

Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions.

Interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 are major hematopoietic cytokines produced by activated T cells and exhibit similar biologic activities by signaling through a common receptor subunit (beta c). Mice lacking beta c show a pulmonary alveolar proteinosis-like disease and reduced numbers of peripheral eosinophils, which are explained by the lack of GM-CSF and IL-5 function, respectively. However, beta c-deficient hematopoietic cells do respond to IL-3 normally, probably through an additional beta subunit of the IL-3 receptor (beta IL3) that is present in the mouse. Thus, almost normal hematopoiesis in beta c-deficient mice may be caused by functional redundancy between IL-3 and GM-CSF. To clarify the role of the entire IL-3/GM-CSF/IL-5 system in hematopoiesis in vivo, we crossed the beta c mutant mice with mice deficient for IL-3 ligand to generate mice lacking the entire IL-3/GM-CSF/IL-5 functions. The double-mutant mice were apparently normal and fertile. The severity of the lung pathology in the beta c/IL-3 double-mutant mice showed normal hemodynamic parameters except for reduced numbers of eosinophils and the lack of eosinophilic response to parasites, which were also found in beta c mutant mice. The immune response of the beta c/IL-3 double-mutant mice to Listeria mono-cytogenes was normal, as was hematopoietic recovery after administration of the cytotoxic drug, 5-fluorouracil. Although it has been believed that IL-3/GM-CSF/IL-5 produced by activated T cells play a major role in expansion of hematopoietic cells in emergency, our results indicate that the entire function of IL-3/GM-CSF/IL-5 is dispensable for hematopoiesis in emergency as well as in the steady state. Thus, there must be an alternative mechanism to produce blood cells in both situations.

The pulmonary alveolar proteinosis in granulocyte macrophage colony-stimulating factor/interleukins 3/5 beta c receptor-deficient mice is reversed by bone marrow transplantation.

Mice mutant for granulocyte macrophage colony-stimulating factor (GM-CSF) or the common receptor component (beta c) for GM-CSF, interleukin (IL)-3, and IL-5 exhibit a lung disorder similar to human pulmonary alveolar proteinosis, a rare disease with congenital, infantile, and adult forms. Bone marrow transplantation and hematopoietic reconstitution of beta c mutant mice with wild-type bone marrow reversed the established disease state in the lungs, defining this disease as hematopoietic in nature. It is likely that the disease involves alveolar macrophages, as donor myeloid cell engraftment into the lungs of mutant recipient mice correlated with reverting both the disease and an abnormal macrophage morphology seen in the lungs of affected animals. Recombination Activating Gene-2 mutant donor bone marrow, which lacks the potential to develop lymphocytes, reversed the pathology in the lungs to the same extent as whole bone marrow. These data establish that certain lung disorders, if of cell-autonomous hematopoietic origin, can be manipulated by bone marrow transplantation.

Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response, while beta IL3 receptor-deficient mice are normal.

The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an additional IL-3 beta subunit (beta IL3). We have independently generated mice carrying null mutations of each molecule. beta c mutant bone marrow showed no response to GM-CSF or IL-5, whereas IL-3 stimulation of beta c or beta IL3 mutant bone marrow was normal. beta c mutant mice showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis, and also exhibited low basal numbers of eosinophils. Infection of beta c mutant mice by Nippostrongylus brasiliensis resulted in the absence of blood and lung eosinophilia. Animals repopulated with beta c mutant bone marrow cells showed slower leukocyte recovery and reduced eosinophil numbers. These data define the role of beta c in vivo, and show a phenotype that is likely to be the cumulative effect of loss of GM-CSF and IL-5 stimulation.