The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

A prominent pathway of transforming growth factor (TGF)-␤ signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-␤1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-␤1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-␤1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [ 3 H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-␤1. Lack of Smad2 or Smad3 expression did not affect TGF-␤1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-␤1. Moreover, TGF-␤1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-␤1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-␤-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element) 4 -Lux reporter by TGF-␤1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-␤1 signaling.

Since 1992, there has been growing evidence that the bioactive phospholipid lysophosphatidic acid (LPA), whose amounts are increased upon tissue injury, activates primary nociceptors resulting in neuropathic pain. The TRPV1 ion channel is expressed in primary afferent nociceptors and is activated by physical and chemical stimuli. Here we show that in control mice LPA produces acute pain-like behaviors, which are substantially reduced in Trpv1-null animals. Our data also demonstrate that LPA activates TRPV1 through a unique mechanism that is independent of G protein-coupled receptors, contrary to what has been widely shown for other ion channels, by directly interacting with the C terminus of the channel. We conclude that TRPV1 is a direct molecular target of the pain-producing molecule LPA and that this constitutes, to our knowledge, the first example of LPA binding directly to an ion channel to acutely regulate its function.

Bioactive phospholipids, which include sphingosine-1-phosphate,lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid phosphate phosphatases (LPPs). Here we show that mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature. A subset of embryos also show a shortening of the anterior-posterior axis and frequent duplication of axial structures that are strikingly similar to the phenotypes associated with axin deficiency,a critical regulator of Wnt signaling. Loss of LPP3 results in a marked increase in β-catenin-mediated TCF transcription, whereas elevated levels of LPP3 inhibit β-catenin-mediated TCF transcription. LPP3 also inhibits axis duplication and leads to mild ventralization in Xenopusembryo development. Although LPP3 null fibroblasts show altered levels of bioactive phospholipids, consistent with loss of LPP3 phosphatase activity, mutant forms of LPP3, specifically lacking phosphatase activity, were able to inhibit β-catenin-mediated TCF transcription and also suppress axis duplication, although not as effectively as intact LPP3. These results reveal that LPP3 is essential to formation of the chorio-allantoic placenta and extra-embryonic vasculature. LPP3 also mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway. The exact biochemical roles of LPP3 phosphatase activity and its undefined effect on β-catenin-mediated TCF transcription remain to be determined.

Imprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the -sarcoglycan gene (Sgce) and Zac1 (Lot1) are both paternally expressed imprinted genes. They were identified in a subtractive screen for imprinted genes using a cDNA library made from novel parthenogenetic and wild-type fibroblast lines. Sgce is a component of the dystrophin-sarcoglycan complex, Zac1 is a nuclear protein inducing growth arrest and/or apoptosis, and Zac1 is a potential tumor suppressor gene. Sgce and Zac1 are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1 is biallelic in the liver and Sgce is weakly expressed from the maternal allele in the brain. Sgce and Zac1 are broadly expressed in embryos, with Zac1 being highly expressed in the liver primordium, the umbilical region, and the neural tube. Sgce, however, is strongly expressed in the allantoic region on day 9.5 but becomes more widely expressed throughout the embryo by day 11.5. Sgce is located at the proximal end of mouse chromosome 6 and is a candidate gene for embryonic lethality associated with uniparental maternal inheritance of this region. Zac1 maps to the proximal region of chromosome 10, identifying a new imprinted locus in the mouse, homologous with human chromosome 6q24-q25. In humans, unipaternal disomy for this region is associated with fetal growth retardation and transient neonatal diabetes mellitus. In addition, loss of expression of ZAC has been described for a number of breast and ovarian carcinomas, suggesting that ZAC is a potential tumor suppressor gene.

SMAD2 is a member of the transforming growth factor ␤ and activin-signaling pathway. To examine the role of Smad2 in postgastrulation development, we independently generated mice with a null mutation in this gene. Smad2-deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior-posterior (A-P) axis. Expression of the homeobox gene Hex (the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in Smad2-deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues, Smad2-deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that Smad2 expression is required at several stages during embryogenesis. T he transforming growth factor ␤ (TGF-␤) superfamily is a group of signaling molecules that play essential roles in development and tissue homeostasis in vertebrates and invertebrates (1-3). Members of this family include the TGF-␤s, activins, and bone morphogenetic proteins.A family of proteins known as SMADs has been shown to function downstream of TGF family type I receptors (reviewed in ref. 4). The TGF-␤ and activin type I receptors interact with SMAD2 and the closely related SMAD3 (5-9). After being phosphorylated by type I receptors, pathway-restricted SMADs associate with SMAD4 (10), translocate to the nucleus, and activate, in conjunction with several specific transcription factors, their respective target genes (4, 11).The SMAD2 protein consists of three domains (4), the Mad homology domain 1 (MH1) and the MH2 domain that harbors the DNA-binding site (12). The third linker region domain connects the MH1 and MH2 domains, is phosphorylated, and may serve as a molecular hinge. The MH2 domain interacts with the type I receptors and is phosphorylated on ligand activation. The SMAD2-type I receptor interaction is also regulated by a newly discovered protein named SARA (13). The MH2 domain also exhibits transactivation properties.The human SMAD2 gene is located on chromosome 18q21, in close proximity to the tumor suppressors DCC and SMAD4͞DPC4 (14-16). Mutations that disrupt the function of SMAD2 have been identified in colorectal cancers (16,17) and lung cancer (18), suggesting its role as a tumor suppressor.Smad2 is expressed almost ubiquitously and has an important role in early development (19)(20)(21)(22). Targeted inactivation of Smad2 resulted in early lethality before gastrulation. Though all these studies showed that Smad2-deficient embryos die because of gastrulation failure, two studies did not describe mesoderm formation (21,22), whereas the other reported transient mesoderm induction but failur...

Lipid phosphate phosphatase 3 in endothelial and epithelial cells promotes efficient T cell emigration from the thymus to the periphery.

Structured Abstract Objective Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein-coupled receptor-mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in development and human disease. Approach and results We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek (Tie2) promoter-mediated inactivation of Ppap2b resulted in embryonic lethality due to vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tie2 promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3-deficiency led to significant increases in vascular permeability at baseline, and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein-coupled receptor-dependent LPA signaling. Conclusions Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.