Smad expression during kidney development.
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Signaling by the transforming growth factor (TGF)-beta superfamily is important during kidney development. Here, we describe the spatial and temporal expression patterns of the Smads, the transcription factors that translate TGF- signals into gene expression. RT-PCR data and in situ hybridization analysis showed that the receptor-regulated (R) Smads (Smad1, -2, -3, -5, and -8), the common partner Smad (Smad4), and the inhibitory (I) Smads (Smad6 and -7) were all expressed during mouse kidney development from embryonic day 12 until the end of nephrogenesis at postnatal day 15. Each Smad had a distinct spatial distribution. All were expressed by mesenchymal cells in the nephrogenic zone and were downregulated once these cells began to epithelialize. The common partner Smad, Smad4, was present in uninduced mesenchymal cells and at ureteric bud tips. The bone morphogenetic-responsive R-Smads, Smad1, -5, and -8, were mainly expressed in the nephrogenic zone, whereas the TGF-- responsive R-Smads were predominantly noted in the medullary interstitium. Expression of the I-Smad Smad7 was also seen in mesenchymal cells in the interstitium. Based on the observed patterns of expression, we speculate that individual or combinations of Smads may play specific roles in cell-fate determination during kidney development. (+info)
SMAD5 gene expression, rearrangements, copy number, and amplification at fragile site FRA5C in human hepatocellular carcinoma.
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Signaling by the transforming growth factor (TGF)-family members is transduced from the cell surface to the nucleus by the Smad group of intracellular proteins. Because we detected alterations on the long arm of chromosome 5, we examined the status of the SMAD5 gene in human hepatocellular carcinoma (HCC) cell lines and primary HCC. In 16 cell lines, chromosome alterations of chromosome 5 were observed in nine cell lines by fluorescence in situ hybridization (FISH), and an increase in SMAD5 gene copy number relative to the ploidy level was found in eight lines. The breakpoints in unbalanced translocations and deletions frequently occurred near the SMAD5 locus, but apparently did not cause loss of SMAD5. In one cell line, where comparative genomic hybridization showed DNA copy number gain confined to the region 5q31, we detected by FISH high-level amplification of the SMAD5 gene located within the fragile site FRA5C. Semiquantitative polymerase chain reaction did not reveal changes in SMAD5 DNA levels in 15 of 17 primary HCC specimens. In 17 HCC cell lines, SMAD5 mRNA levels were either maintained or upregulated by an increase in gene dosage or another mechanism. Collectively, our results show that SMAD5 undergoes copy number gain and increased expression, rather than loss of expression, and therefore suggest that this gene does not act as a tumor-suppressor gene in HCC. The Hep-40 HCC cell line with high-level amplification and significant overexpression of SMAD5 may be useful in studying the interaction of SMAD5 with other genes. (+info)
Overexpression of Smurf1 negatively regulates mouse embryonic lung branching morphogenesis by specifically reducing Smad1 and Smad5 proteins.
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Early embryonic lung branching morphogenesis is regulated by many growth factor-mediated pathways. Bone morphogenetic protein 4 (BMP4) is one of the morphogens that stimulate epithelial branching in mouse embryonic lung explant culture. To further understand the molecular mechanisms of BMP4-regulated lung development, we studied the biological role of Smad-ubiquitin regulatory factor 1 (Smurf1), an ubiquitin ligase specific for BMP receptor-regulated Smads, during mouse lung development. The temporo-spatial expression pattern of Smurf1 in mouse embryonic lung was first determined by quantitative real-time PCR and immunohistochemistry. Overexpression of Smurf1 in airway epithelial cells by intratracheal introduction of recombinant adenoviral vector dramatically inhibited embryonic day (E) 11.5 lung explant growth in vitro. This inhibition of lung epithelial branching was restored by coexpression of Smad1 or by addition of soluble BMP4 ligand into the culture medium. Studies at the cellular level show that overexpression of Smurf1 reduced epithelial cell proliferation and differentiation, as documented by reduced PCNA-positive cell index and by reduced mRNA levels for surfactant protein C and Clara cell protein 10 expression. Further studies found that overexpression of Smurf1 reduced BMP-specific Smad1 and Smad5, but not Smad8, protein levels. Thus overexpression of Smurf1 specifically promotes Smad1 and Smad5 ubiquitination and degradation in embryonic lung epithelium, thereby modulating the effects of BMP4 on embryonic lung growth. (+info)
A hierarchical order of factors in the generation of FLK1- and SCL-expressing hematopoietic and endothelial progenitors from embryonic stem cells.
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The receptor tyrosine kinase FLK1 and the transcription factor SCL play crucial roles in the establishment of hematopoietic and endothelial cell lineages in mice. We have previously used an in vitro differentiation model of embryonic stem (ES) cells and demonstrated that hematopoietic and endothelial cells develop via sequentially generated FLK1(+) and SCL(+) cells. To gain a better understanding of cellular and molecular events leading to hematopoietic specification, we examined factors necessary for FLK1(+) and SCL(+) cell induction in serum-free conditions. We demonstrate that bone morphogenetic protein (BMP) 4 was required for the generation of FLK1(+) and SCL(+) cells, and that vascular endothelial growth factor (VEGF) was necessary for the expansion and differentiation of SCL-expressing hematopoietic progenitors. Consistently, Flk1-deficient ES cells responded to BMP4 and generated TER119(+) and CD31(+) cells, but they failed to expand in response to VEGF. The Smad1/5 and map kinase pathways were activated by BMP4 and VEGF, respectively. The overexpression of SMAD6 in ES cells resulted in a reduction of FLK1(+) cells. In addition, a MAP kinase kinase 1 specific inhibitor blocked the expansion of SCL(+) cells in response to VEGF. Finally, VEGF mediated expansion of hematopoietic and endothelial cell progenitors was inhibited by TGFbeta1, but was augmented by activin A. Our studies suggest that hematopoietic and endothelial commitment from the mesoderm occurs via BMP4-mediated signals and that expansion and/or differentiation of such progenitors is achieved by an interplay of VEGF, TGFbeta1 and activin A signaling. (+info)
Menin is required for bone morphogenetic protein 2- and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2.
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Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late-stage differentiation. (+info)
Bmp2 antagonizes sonic hedgehog-mediated proliferation of cerebellar granule neurones through Smad5 signalling.
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During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. We have looked for signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurones. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurones. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. We propose a model in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme. (+info)
Spatio-temporal activation of Smad1 and Smad5 in vivo: monitoring transcriptional activity of Smad proteins.
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Signaling by bone morphogenetic proteins is essential for a wide variety of developmental processes. Receptor-regulated Smad proteins, Smads 1 and 5, are intracellular mediators of bone morphogenetic protein signaling. Together with Smad4, these proteins translocate to the nucleus and modulate transcription by binding to specific sequences on the promoters of target genes. We sought to map transcriptional Smad1/5 activity in development by generating embryonic stem cell lines carrying a Smad1/5-specific response element derived from the Id1 promoter coupled to beta-galactosidase or luciferase as reporters. Three independent lines (BRE-lac1, BRE-lac2 and BRE-luc) have shown the existence of an autocrine bone morphogenetic protein signaling pathway in mouse embryonic stem cells. Reporter activity was detected in chimeric embryos, suggesting sensitivity to physiological concentrations of bone morphogenetic protein. Reporter activity in embryos from transgenic mouse lines was detected in tissues where an essential role for active bone morphogenetic protein signaling via Smads 1 or 5 had been previously established. We have thus generated, for the first time, an in vivo readout for studying the role of Smad1/5-mediated transcriptional activity in development. (+info)
BMP4 and Madh5 regulate the erythroid response to acute anemia.
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Acute anemia initiates a systemic response that results in the rapid mobilization and differentiation of erythroid progenitors in the adult spleen. The flexed-tail (f) mutant mice exhibit normal steady-state erythropoiesis but are unable to rapidly respond to acute erythropoietic stress. Here, we show that f/f mutant mice have a mutation in Madh5. Our analysis shows that BMP4/Madh5-dependent signaling, regulated by hypoxia, initiates the differentiation and expansion of erythroid progenitors in the spleen. These findings suggest a new model where stress erythroid progenitors, resident in the spleen, are poised to respond to changes in the microenvironment induced by acute anemia. (+info)