Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development.

BACKGROUND: Smad4 mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer. Conditional rescue experiments demonstrate that bypassing the primary requirement for Smad4 in the extra-embryonic endoderm allows the epiblast to gastrulate. Smad4-independent TGF-beta signals are thus sufficient to promote mesoderm formation and patterning. To further analyse essential Smad4 activities contributed by the extra-embryonic tissues, and characterise Smad4 dependent pathways in the early embryo, here we performed transcriptional profiling of Smad4 null embryonic stem (ES) cells and day 4 embryoid bodies (EBs). RESULTS: Transcripts from wild-type versus Smad4 null ES cells and day 4 EBs were analysed using Illumina arrays. In addition to several known TGF-beta/BMP target genes, we identified numerous Smad4-dependent transcripts that are mis-expressed in the mutants. As expected, mesodermal cell markers were dramatically down-regulated. We also observed an increase in non-canonical potency markers (Pramel7, Tbx3, Zscan4), germ cell markers (Aire, Tuba3a, Dnmt3l) as well as early endoderm markers (Dpp4, H19, Dcn). Additionally, expression of the extracellular matrix (ECM) remodelling enzymes Mmp14 and Mmp9 was decreased in Smad4 mutant ES and EB populations. These changes, in combination with increased levels of laminin alpha1, cause excessive basement membrane deposition. Similarly, in the context of the Smad4 null E6.5 embryos we observed an expanded basement membrane (BM) associated with the thickened endoderm layer. CONCLUSION: Smad4 functional loss results in a dramatic shift in gene expression patterns and in the endodermal cell lineage causes an excess deposition of, or an inability to breakdown and remodel, the underlying BM layer. These structural abnormalities probably disrupt reciprocal signalling between the epiblast and overlying visceral endoderm required for gastrulation.

Global expression profiling of sex cord stromal tumors from Men1 heterozygous mice identifies altered TGF-beta signaling, decreased Gata6 and increased Csf1r expression.

Heterozygous disruption of the Men1 gene predisposes mice to the development of multiple endocrine tumors, accurately mimicking the human MEN1 cancer predisposition syndrome. Additionally, Men1(+/-) mice frequently develop sex cord adenomas. The mechanism underlying the susceptibility of these mice to sex cord tumor development has not been fully determined, but data suggest it may involve transcriptional regulation of key growth promoting/repressing genes. To identify potential menin-regulated genes that may be important for tumor suppression in sex cord cells, we compared the global gene expression profiles of testis and ovary adenomas with other endocrine tumors of the pancreas and pituitary from Men1 heterozygous mice and with control tissues. Gonadal tumors clustered separately from pancreas and pituitary tumors with only a few genes (e.g., Cdkn2c) commonly dysregulated in all tumor types. Testis and ovary tumors displayed a higher level of transcriptional similarity to each other than they did to their respective control tissues. Among genes that had decreased expression in tumors was significant over-representation of genes associated with the TGF-beta, hedgehog and Wnt signaling, indicating that loss of menin function affects these pathways at the level of transcription. Aberrant protein expression in Leydig and granulosa cells of 2 transcriptionally dysregulated gene products, Gata6 and Csf1r were confirmed by immunohistochemistry. We propose that sex cord tumor susceptibility in Men1(+/-) mice involves deregulated cell proliferation due to dysregulation of multiple cell growth regulating genes including: reduced Cdkn2c transcription, loss of TGF-beta pathway tumor suppressor function (e.g., Gata6) and transcriptional activation of Csf1r.

Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling.

Smad4 in partnership with R-Smads (receptor-regulated Smads) activates TGF-beta (transforming growth factor-beta)-dependent signalling pathways essential for early mouse development. Smad4 null embryos die shortly after implantation due to severe defects in cell proliferation and visceral endoderm differentiation. In the basal state, Smad4 undergoes continuous shuttling between the cytoplasm and the nucleus due to the combined activities of an N-terminal NLS (nuclear localization signal) and an NES (nuclear export signal) located in its linker region. Cell culture experiments suggest that Smad4 nucleocytoplasmic shuttling plays an important role in TGF-beta signalling. In the present study we have investigated the role of Smad4 shuttling in vivo using gene targeting to engineer two independent mutations designed to eliminate Smad4 nuclear export. As predicted this results in increased levels of Smad4 in the nucleus of homozygous ES cells (embryonic stem cells) and primary keratinocytes, in the presence or absence of ligand. Neither mutation affects Smad4 expression levels nor its ability to mediate transcriptional activation in homozygous cell lines. Remarkably mouse mutants lacking the Smad4 NES develop normally. Smad4 NES mutants carrying one copy of a Smad4 null allele also fail to display developmental defects. The present study clearly demonstrates that Smad4 nucleocytoplasmic shuttling is not required for embryonic development or tissue homoeostasis in normal, healthy adult mice.

Conditional inactivation of the MEN1 gene leads to pancreatic and pituitary tumorigenesis but does not affect normal development of these tissues.

Mutations of the MEN1 gene, encoding the tumor suppressor menin, predispose individuals to the cancer syndrome multiple endocrine neoplasia type 1, characterized by the development of tumors of the endocrine pancreas and anterior pituitary and parathyroid glands. We have targeted the murine Men1 gene by using Cre recombinase-loxP technology to develop both total and tissue-specific knockouts of the gene. Conditional homozygous inactivation of the Men1 gene in the pituitary gland and endocrine pancreas bypasses the embryonic lethality associated with a constitutional Men1(-/-) genotype and leads to beta-cell hyperplasia in less than 4 months and insulinomas and prolactinomas starting at 9 months. The pituitary gland and pancreas develop normally in the conditional absence of menin, but loss of this transcriptional cofactor is sufficient to cause beta-cell hyperplasia in some islets; however, such loss is not sufficient to initiate pituitary gland tumorigenesis, suggesting that additional genetic events are necessary for the latter.

A crucial role for bone morphogenetic protein-Smad1 signalling in the DNA damage response.

DNA damage and the elicited cellular response underlie the etiology of tumorigenesis and ageing. Yet, how this response integrates inputs from cells' environmental cues remains underexplored. Here we report that the BMP-Smad1 pathway, which is essential for embryonic development and tissue homeostasis, has an important role in the DNA damage response and oncogenesis. On genotoxic stress, Atm phosphorylates BMPs-activated Smad1 in the nucleus on S239, which disrupts Smad1 interaction with protein phosphatase PPM1A, leading to enhanced activation and upregulation of Smad1. Smad1 then interacts with p53 and inhibits Mdm2-mediated p53 ubiquitination and degradation to regulate cell proliferation and survival. Enhanced Smad1 S239 phosphorylation, and Smad1 mutations causing S239 substitution were detected in oesophageal and gastric cancer samples, respectively. These findings suggest that BMP-Smad1 signalling participates in the DNA damage response via the Atm-p53 pathway, thus providing a molecular mechanism whereby BMP-Smad1 loss-of-function leads to tumorigenesis, for example, juvenile polyposis and Cowden syndromes.

Global expression profiling of murine MEN1-associated tumors reveals a regulatory role for menin in transcription, cell cycle and chromatin remodelling.

Although the identification of menin-interacting partners and other evidence support a role for menin, the multiple endocrine neoplasia type 1 gene (MEN1) product, in regulating gene expression, little is known about the cellular pathways dysregulated by menin loss during tumorigenesis. The mouse models of MEN1 accurately mimic the human syndrome and provide an opportunity to assess the transcriptional effects of Men1 deletion in different endocrine tumor types to identify common pathway aberrations underlying tumorigenesis in MEN1-affected tissues. We compared the global gene expression profiles of pituitary adenomas and pancreatic islet tumors with control tissues from wild-type littermates. Amongst the 551 differentially expressed genes was significant over-representation of genes associated with chromatin remodelling, transcription and cell cycling, including some genes known to encode menin-binding partners, e.g., Rhox5 and Mll1. Consistent with increased cell-cycle transition from G1 to S phase was an elevation of Cdc7 expression in the tumors, which was confirmed by qRT-PCR using independent samples. In support of previous findings in islet tumors, we found down-regulation of the cell-cycle regulator, p18, in both the pancreatic islet and pituitary adenomas, suggesting that reduced p18 levels may be important for Men1-related tumorigenesis in multiple tissues. Surprisingly, we identified increased p16 transcript in pancreatic islet and pituitary tumors. This was accompanied by increased cytoplasmic localization p16 protein in tumor cells. The specific genes and general pathways we have found to be commonly dysregulated in MEN1 tumors, provide a platform for determining their roles in endocrine tumorigenesis.

Broad tumor spectrum in a mouse model of multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an inherited cancer predisposition syndrome typified by development of tumors in parathyroid, pituitary and endocrine pancreas, as well as less common sites including both endocrine and nonendocrine organs. Deletion or mutation of the tumor suppressor gene MEN1 on chromosome 11 has been identified in many cases of MEN1 as well as in sporadic tumors. The molecular biology of menin, the protein encoded by MEN1, remains poorly understood. Here we describe a mouse model of MEN1 in which tumors were seen in pancreatic islets, pituitary, thyroid and parathyroid, adrenal glands, testes and ovaries. The observed tumor spectrum therefore includes types commonly seen in MEN1 patients and additional types. Pancreatic pathology was most common, evident in over 80% of animals, while other tumor types developed with lower frequency and generally later onset. Tumors of multiple endocrine organs were observed frequently, but progression to carcinoma and metastasis were not evident. Tumors in all sites showed loss of heterozygosity at the Men1 locus, though the frequency in testicular tumors was only 36%, indicating that a different molecular mechanism of tumorigenesis occurs in those Leydig tumors that do not show loss of the normal Men1 allele. Menin expression was below the level of detection in ovary, thyroid and testis, but loss of nuclear menin immunoreactivity was observed uniformly in all pancreatic islet adenomas and in some hyperplastic islet cells, suggesting that complete loss of Men1 is a critical point in islet tumor progression in this model.

Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus.

The cellular function of the menin tumor suppressor protein, product of the MEN1 gene mutated in familial multiple endocrine neoplasia type 1, has not been defined. We now show that menin is associated with a histone methyltransferase complex containing two trithorax family proteins, MLL2 and Ash2L, and other homologs of the yeast Set1 assembly. This menin-associated complex methylates histone H3 on lysine 4. A subset of tumor-derived menin mutants lacks the associated histone methyltransferase activity. In addition, menin is associated with RNA polymerase II whose large subunit carboxyl-terminal domain is phosphorylated on Ser 5. Men1 knockout embryos and cells show decreased expression of the homeobox genes Hoxc6 and Hoxc8. Chromatin immunoprecipitation experiments reveal that menin is bound to the Hoxc8 locus. These results suggest that menin activates the transcription of differentiation-regulating genes by covalent histone modification, and that this activity is related to tumor suppression by MEN1.