Since the discovery that the TGF-β signalling molecule Nodal and its downstream effector Pitx have a parallel role in establishing asymmetry between molluscs and deuterostomes the debate over the degree to which this signalling pathway is conserved across the Bilateria as a whole has been ongoing. Further taxon sampling is critical to understand the evolution and divergence of this signalling pathway in animals. Using genome and transcriptome mining we confirmed the presence of nodal and Pitx in a range of additional animal taxa for which their presence has not yet been described. In situ hybridization was used to show the embryonic expression of these genes in brachiopods and planarians. We show that both nodal and Pitx genes are broadly conserved across the Spiralia, and nodal likely appeared in the Bilaterian stem lineage after the divergence of the Acoelomorpha. Furthermore, both nodal and Pitx mRNA appears to be expressed in an asymmetric fashion in the brachiopod Terebratalia transversa. No evidence for the presence of a Lefty ortholog could be found in the non-deuterostome genomic resources examined. Nodal expression is asymmetric in a number of spiralian lineages, indicating a possible ancestral role of the Nodal/Pitx cascade in the establishment of asymmetries across the Bilateria.

To monitor human embryonic stem cell (hESC) self-renewal without differentiation, we used quantitative RT-PCR to study a selection of hESC genes, including markers for self-renewal, commitment/differentiation, and members of the TGF-beta superfamily and DAN gene family. Indeed, low commitment/differentiation gene expression, together with a significant self-renewal gene expres sion, provides a better pluripotency index than self-renewal genes alone. We demonstrate that matrices derived from human mesenchymal stem cells (hMSCs) can advantageously replace murine embryonic fibroblasts (MEF) or hMSC feeders. Moreover, a xenofree molecularly-defined SBX medium, containing a synthetic lipid carrier instead of albumin, can replace SR medium. The number of selected differentiation genes expressed by hESCs in these culture conditions was significantly lower than those expressed on MEF feeders in SR medium. In SBX, the positive effect of a non-physiological concentration of activin A (10-30 ng/mL) to reduce differentiation during self-renewal could also be obtained by physiological concentrations of TGF-beta(100-300 pg/mL). In contrast, these TGF-beta concentrations added to activin favored differentiation as previously observed with TGF-beta concentrations of 1 ng/mL or more. Compared to SR-containing medium, SBX medium promoted down-regulation of CER1 and LEFTIES and up-regulation of GREM1. Thus these genes better control self-renewal and pluripotency and prevent differentiation. A strategy is proposed to analyze, in more physiological, xenofree, molecularly-defined media and matrices, the numerous genes with still unknown functions controlling hESCs or human-induced pluripotent stem cells (iPS).

Various matrix metalloproteinases (MMPs) participate in the menstrual breakdown of the human endometrium. MMP-9/gelatinase B is proposed as a major factor because it degrades many extracellular matrix constituents, including in the vasculature. Although globally under ovarian steroids control, endometrial MMP-9 seems expressed differently than other MMPs, and conflicting publications prevent a clear understanding of its regulation. We therefore quantified MMP-9 expression in the cycling human endometrium, defined its localization, and analyzed its regulation by estradiol and progesterone and by LEFTY-A/endometrial bleeding-associated factor in explant cultures. In fresh tissues, a major increase in MMP-9 mRNA expression occurred at menstruation, after a larger increase in LEFTY-A mRNA. MMP-9 was immunodetected in all cell types throughout the cycle, especially in foci of stromal cells during menstruation. MMP-9 synthesis by these cells was confirmed in cultured explants. In proliferative explants, ovarian steroids slightly decreased MMP-9 mRNA. They had no consistent effect on MMP-9 release in culture medium but strongly inhibited proMMP-9 activation. Addition of recombinant LEFTY-A to explants induced MMP-9 in most samples, a response prevented by ovarian steroids. We propose that endometrial MMP-9 activity is overall controlled by the ovarian steroids and locally adjusted through a network of modulators, including LEFTY-A.

BACKGROUND

Nodal (nodal growth differentiation factor) and its inhibitor Lefty (left right determination factor), which are ligands of the TGF (transforming growth factor) β superfamily, are responsible for the determination of left-right asymmetry in vertebrates. Nodal/Lefty signaling has been suggested to play a role in the development of metastatic melanoma and breast cancer. However, it remains unclear whether this pathway is also involved in human pancreatic ductal adenocarcinoma (PDAC).

METHODS

Pancreatic cancer patient specimens with clinical data (n = 54) were used to investigate the clinical significance of Nodal-Lefty signaling. A set of in vitro assays were carried out in a human pancreatic cancer cell line (Colo-357) to assess the functional relevance of Nodal-Lefty signaling.

RESULTS

Nodal was absent in the human normal pancreas, while Lefty was present in islet cells. Though Nodal and Lefty expression were found in cancer cells at various expression levels, the cancer-associated tubular complexes were particularly positive for Lefty. Survival analysis revealed that high expression of Nodal correlated with reduced patient survival (median survival 17.8 vs 33.0 months, p = 0.013). Cultured pancreatic cancer cell lines expressed Nodal and Lefty at different levels. In vitro functional assays revealed that treatment with human recombinant Nodal inhibited cell growth and increased invasion of Colo-357 pancreatic cancer cells whereas no effect was found upon treatment with recombinant Lefty.

CONCLUSIONS

Nodal-Lefty signaling might be involved in the pathogenesis of PDAC as Nodal expression marks a subtype of PDAC with unfavorable prognosis.

1. The present study examined the role of C-phycocyanin (C-pc) in relation to growth factors and cell migration during wound healing. 2. Histological and biochemical studies showed that C-pc scaffold significantly (P < 0.01) increased hydroxyl proline, total hexamine and protein content, and decreased uronic acid content in the wound tissues during a time course study in newly formed skin. 3. Reverse transcription polymerase chain reaction array of mouse growth factors in wound tissue showed overexpression (up to 10-fold) of growth factors, such as Cxcl12, Fgf18, Lefty 1, Lefty 2, Rabep 1 and Zip91, and downregulation (up to -10-fold) of Amh, Bmp 7 and Nodal genes in a 6-day period in C-pc treated groups. Also, Csf 3, Fgf 22, Mdk, Igf 2, transforming growth factor (TGF)-α 1 and interleukin (IL)-1β showed an upregulation of more than 30-fold than the control groups. TGF-β subfamily cytokine growth factors, such as Bmp 2, 4 and 8b, and other growth factors, such as Cxcl 1, showed the highest activity on day 3, showing a transient type of regulation. Western blot analysis showed a positive correlation between gene activity and protein expressions of Bmp 8b, Bmp4, Bmp2 and Cxcl 1. Day 6 in the C-pc group showed the highest csf3 and IL-1β expression. 4. C-pc had no direct effect on keratinocyte migration. However, keratinocytes that were co-cultured with fibroblasts showed a significantly higher rate of migration in the presence of C-pc, showing an indirect effect of C-pc on keratinocyte migration. 5. In conclusion, biodegradable C-pc scaffold might help to serve as an alternate scaffold material for wound healing.

Nuclear localization of beta-catenin is most likely the first step of embryonic axis formation or embryonic cell specification in a wide variety of animal groups. Therefore, the elucidation of beta-catenin target genes is a key research subject in understanding the molecular mechanisms of the early embryogenesis of animals. In Ciona savignyi embryos, nuclear accumulation of beta-catenin is the first step of endodermal cell specification. Previous subtractive hybridization screens of mRNAs between beta-catenin-overexpressed embryos and nuclear beta-catenin-depleted embryos have resulted in the identification of beta-catenin downstream genes in Ciona embryos. In the present study, I characterize seven additional beta-catenin downstream genes, Cs-cadherinII, Cs-protocadherin, Cs-Eph, Cs-betaCD1, Cs-netrin, Cs-frizzled3/6, and Cs-lefty/antivin. All of these genes were expressed in vegetal blastomeres between the 16-cell and 110-cell stages, although their spatial and temporal expression patterns were different from one another. In situ hybridizations and real-time PCR revealed that the expression of all of these genes was up-regulated in beta-catenin-overexpressed embryos, and down-regulated in beta-catenin-suppressed embryos. Therefore, the accumulation of beta-catenin in the nuclei of vegetal blastomeres activates various vegetally expressed genes with potentially important functions in the specification of these cells.

OBJECTIVE

The objectives of the present study were (i) to identify a novel tumor suppressor gene whose expression level was regulated by transforming growth factor (TGF-β) and (ii) to evaluate the effect of Ras/MEK/ERK signaling on TGF-β-dependent Lefty up-regulation.

METHODS

Human pancreatic cancer cell lines were used. The effect of Ras/MEK/ERK pathway on TGF-β-mediated Lefty up-regulation was tested by adding K-ras small interfering RNA, MEK inhibitor U0126, or extracellular signal-regulated kinase (ERK) inhibitor LY294002.

RESULTS

Transforming growth factor β upregulated Lefty messenger RNA levels within 6 of the 7 cell lines. Lefty exerts an antagonistic effect against the tumor-promoting molecule, Nodal, as recombinant Lefty suppressed Nodal-mediated proliferation. Interestingly, inhibition of the Ras/MEK/ERK pathway dramatically enhanced TGF-mediated Lefty up-regulation, suggesting that Ras/MEK/ERK signaling suppresses TGF-β-Lefty pathway.

CONCLUSIONS

Our data suggest that Lefty is a novel TGF-β target molecule that mediates growth inhibition of pancreatic cancer cells. In addition, activation of the Ras/MEK/ERK pathway serves as a mechanism by which pancreatic cancer escapes from growth inhibition by the TGF-β-Lefty axis. The results imply a novel therapeutic strategy for pancreatic cancer, that is, combination treatment with Ras/MEK/ERK inhibitors and TGF-β.

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-betabeta superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-betaLefty A or control vectors and stimulated with 10 ng/ml TGF-betabetaLefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-betaLefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.

During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.

To identify proteins involved in ovarian clear cell carcinoma (OCCCa), shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples of ovarian carcinoma. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY), a novel member of the transforming growth factor-β superfamily. In 143 cases of ovarian epithelial carcinoma including 99 OCCCas and 44 non-OCCCas, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas, with the mRNA expression of LEFTYLEFTYLEFTYLEFTYLEFTY score relative to those with a low score. These findings suggest that LEFTY may be an excellent OCCCa-specific molecular marker, which has anti-tumor effects in altering cell proliferation and cellular susceptibility to apoptosis.

Left-right asymmetric signaling molecules in mammals include three transforming growth factor <em>beta</em> (TGF<em>beta</em>)-related factors, Nodal, <em>Lefty</em>1 and <em>Lefty</em>2. They are all expressed on the left half of developing mouse embryos. Nodal acts as a left-side determinant by transducing signals through Smad and FAST and by inducing Pitx2 expression on the left side. <em>Lefty</em> proteins are antagonists that inhibit Nodal signaling. There are positive and negative transcriptional regulatory loops between nodal and lefty2 genes. Thus, Nodal activates its own gene and lefty2. <em>Lefty</em>2 protein produced then inhibits Nodal signaling and terminates expression of both genes. This feedback mechanism can restrict the range and duration of Nodal signaling in developing embryos.

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.

Cancer and stem cells share the ability to silence tumor suppressors. We focused on Lefty, which encodes one of the most abundant tumor suppressors in embryonic stem (ES) cells and is not expressed in somatic cancer cells. We found that transforming growth factor β (TGF-β) induced demethylation of the Lefty B cytosine-phosphate-guanine (CpG) island and increased Lefty expression (10-200 times) in human pancreatic cancer cells and human liver cancer cells (PLC/PRF/5 and HLF). Expression of Cripto, another important factor in Nodal-Lefty signaling, was not increased after adding TGF-β. We generated reprogrammed cancer cells that revealed high expression of immature marker proteins, high proliferation, and the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, suggesting that these cells may have cancer stem cell-like phenotypes. We investigated Lefty and found that reprogrammed human liver cancer cells (induced pluripotent cancer cells) displayed a much lower ability to express Lefty, although less Lefty B CpG methylation was also observed. We also found that a MEK inhibitor dramatically enhanced Lefty expression in human pancreatic cancers with mutated ras, whereas Lefty B CpG methylation was not decreased. These observations indicate that despite the demethylation of DNA strands in promoter regions of pluripotency-associated genes, including Lefty gene, Lefty expression was not induced well in reprogrammed cells. Of note was the fact that Lefty is abundantly expressed in human ES cells but not in induced pluripotent stem (iPS) cells. We thus think that reprogrammed cancer cells share the mechanism for expression of Lefty with iPS cells. This shared mechanism may contribute to the cancerous transformation of iPS cells.

Both normal and neoplastic tissues have a stroma comprised of fibroblasts which deposit an extracellular matrix (ECM) enriched in collagen. In most normal tissues, the synthesis and breakdown of the ECM is maintained at a low level. However, in normal adult tissues such as endometrium, and in rapidly growing embryonic and neoplastic tissues, there is a significant increase in the synthesis and/or breakdown of ECM. The homeostasis of the ECM is maintained by a molecular repertoire which appears to consist of TGF-beta and lefty. TGF-beta acts as a pro-fibrogenic cytokine by increasing the synthesis of collagen and decreasing the degradation of ECM. Physiologic levels of TGF-beta maintains tissue homeostasis and aberrant over-expression of TGF-beta leads to tissue fibrosis. TGF-beta acts through a core Smad signaling pathway which is initiated by the binding of homo-dimeric TGF-beta protein to two type I and II receptors. The constitutively active receptor type II leads to phosphorylation of receptor type I which, in turn, causes the R-Smads to get phosphorylated. The downstream gene transcriptional activity of this event includes significant increase in connective tissue growth factor (CTGF) and collagen mRNA synthesis which leads to deposition of collagen in tissues. Lefty inhibits Smad2 phosphorylation initiated by TGF-beta or its receptor and prevents CTGF promoter activity driven by TGF-beta. Moreover, lefty inhibits CTGF and collagen mRNA synthesis and increases collagenolysis and elastolysis and as a result of these actions, lefty significantly reduces the amount of collagen deposited in tissues. Thus, TGF-beta and lefty might coordinately participate in the homeostasis of ECM in tissues.

To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, alpha-collagen, procollagen, and LAMPI mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor beta (TGF-beta) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.

Human endometrium exhibits cyclic stromal and glandular remodeling in preparation of embryo implantation. We identified EBAF/LEFTY B as a soluble cytokine of the TGF-β superfamily that is expressed at a low level in human endometrium during the receptivity period, while it is maximally expressed during perimenstrual and menstrual phases. Transfection of cells with EBAF/LEFTY B resulted in expression of a 42 kD protein that was proteolytically processed to release two polypeptides of 34 and 28 kD. EBAF/LEFTY B causes tissue remodeling by induction of collagenolysis by matrix metalloproteases. In a subset of infertile patients, the expression EBAF/LEFTY B was prematurely increased during the implantation window. We showed that induced overexpression of EBAF/LEFTY B in transgenic mice impairs implantation. EBAF/LEFTY B inhibits the expression of key decidual proteins, IGFBP-1 and PRL, through regulation of transcription factors FOXO1 and ETS1. Together, these findings show that during embryonic development, EBAF/LEFTY B plays important roles in decidualization and embryo implantation.

OBJECTIVE

To compare the positions of the aorta relative to vertebral bodies and the potential risk of the aorta impingement for pedicle screw (PS) placement between right-sided and left-sided thoracolumbar/lumbar curves of adolescent idiopathic scoliosis (AIS).

METHODS

Thirty-nine AIS patients with a main thoracolumbar or lumbar curve were recruited. The Lenke's classification was type 5C in all patients. According to the convexity of the thoracolumbar or lumbar curves, the patients were divided into either group R or Group L. The patients in Group R had a main right-sided thoracolumbar/lumbar curve, and the patients in Group L had a main left-sided thoracolumbar/lumbar curve. Axial CT images from T12 to L4 at the midvertebral body level were obtained to evaluate Aorta-vertebra angle (α), Vertebral rotation angle (β), Lefty safety distance (LSD), and Right safety distance (RSD). The risks of the aorta impingement from T12 to L4 were calculated and then compared between the two groups.

RESULTS

The α increased from T12 through L4 in Group R, increased from T12 through L1, and then decreased from L1 through L4 in Group L. The β decreased from T12 through L4 in both groups. The LSD constantly increased from T12 through L4 in Group R, increased from T12 through L3, and then decreased from L3 through L4 in Group L. The RSD increased from T12 through L3 and then decreased from L3 through L4 in both groups. With the increment of the lengths of the simulated screws, the aorta impingement risks were constantly elevated at all levels in both groups. The aorta was at a high risk of impingement from left PS regardless of the diameters of the simulated screws in Group R (80-100 % at T12 and 53.3-100 % at L1). In Group L, the aorta was completely safe when using 35 mm (0 at all levels) PS and at high risks of the aorta impingement on the right side from 45 mm PSs (31.8-72.7 %). In all, the risks of the aorta impingement were mainly from left PS in Group R and from right PS in Group L, and the risk of the aorta impingement from PS placement was generally higher in right thoracolumbar or lumbar curves when compared with that of the left.

CONCLUSIONS

The present study illustrated different changed positions of the aorta relative to vertebrae between thoracolumbar/lumbar curves with different convexities. In right-sided curve, the risks of the aorta impingement were mainly from left PS while in left-sided curves, from right PS. The aorta was more proximal to entry points in right-sided lumbar curve when compared with left-sided curve; thus placing PS carries more risks in right-sided thoracolumbar/lumbar curve. Surgeons should be more cautious when placing PSs on the concave sides of T12 and L1 vertebrae of right-sided thoracolumbar/lumbar curves.

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most aggressive of human brain tumors and has a stunning progression with a mean survival of one year from the date of diagnosis. High cell proliferation, angiogenesis and/or necrosis are histopathological features of this cancer, which has no efficient curative therapy. This aggressiveness is associated with particular heterogeneity of the tumor featuring multiple genetic and epigenetic alterations, but also with implications of aberrant signaling driven by growth factors. The transforming growth factor β (TGFβ) superfamily is a large group of structurally related proteins including TGFβ subfamily members Nodal, Activin, Lefty, bone morphogenetic proteins (BMPs) and growth and differentiation factor (GDF). It is involved in important biological functions including morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing and inflammation. This superfamily is also considered to impact on cancer biology including that of GBM, with various effects depending on the member. The TGFβ subfamily, in particular, is overexpressed in some GBM types which exhibit aggressive phenotypes. This subfamily impairs anti-cancer immune responses in several ways, including immune cells inhibition and major histocompatibility (MHC) class I and II abolishment. It promotes GBM angiogenesis by inducing angiogenic factors such as vascular endothelial growth factor (VEGF), plasminogen activator inhibitor (PAI-I) and insulin- like growth factor-binding protein 7 (IGFBP7), contributes to GBM progression by inducing metalloproteinases (MMPs), "pro-neoplastic" integrins (αvββLefty. Activin promotes GBM cell proliferation while GDF yields immune-escape function. On the other hand, BMPs target GICS and induce differentiation and sensitivity to chemotherapy. This multifaceted involvement of this superfamily in GBM necessitates different strategies in anti-cancer therapy. While suppressing the TGFβ subfamily yields advantageous results, enhancing BMPs production is also beneficial.

Nodal, a secreted signaling protein in the transforming growth factor-beta (TGF-β) superfamily, has established roles in vertebrate development. However, components of the Nodal signaling pathway are also expressed at the maternal-fetal interface and have been implicated in many processes of mammalian reproduction. Emerging evidence indicates that Nodal and its extracellular inhibitor Lefty are expressed in the uterus and complex interactions between the two proteins mediate menstruation, decidualization and embryo implantation. Furthermore, several studies have shown that Nodal from both fetal and maternal sources may regulate trophoblast cell fate and facilitate placentation as both embryonic and uterine-specific Nodal knockout mouse strains exhibit disrupted placenta morphology. Here we review the established and prospective roles of Nodal signaling in facilitating successful pregnancy, including recent evidence supporting a potential link to parturition and preterm birth.

Fibroblast activation and proliferation are important for fibroblast-myofibroblast transdifferentiation, a crucial process in the pathological changes that define renal interstitial fibrosis. The left-right determination factor (Lefty) is an important cytokine of the transforming growth factor (TGF)-β family, with two variants, Lefty-1 and Lefty-2, in mice. Lefty has diverse functions, such as the regulation of embryonic development, the inhibition of TGF-βLefty-1 influences fibroblast activation and proliferation, and consequently prevents fibroblast-myofibroblast transdifferentiation, remains unclear. This study aimed to investigate whether Lefty-1 can attenuate TGF-ββLefty-1 significantly prevented this change in a dose-dependent manner. Further analyses demonstrated decreased proliferating cell nuclear antigen, cyclin D1, collagen I(A1), alpha-smooth muscle actin, and fibronectin expression. Lefty-1 further induced remarkable reductions in TGF-ββLefty-1 had no effect on Smad3, JNK-3, and BMP-5 activation and fibroblast-myofibroblast transdifferentiation. Taken together, these findings indicate that Lefty-1 can alleviate TGF-βLefty-1 may prevent fibroblast-myofibroblast transdifferentiation in part via modulations of Smad3, JNK-3, and BMP-5 activities in the TGF-β/BMP signaling pathway.

Dynamic spatiotemporal expression of the nodal gene and its orthologs is involved in the dose-dependent induction and patterning of mesendoderm during early vertebrate embryogenesis. We report loss-of-function studies that define a high degree of synergistic negative regulation on the Xenopus nodal-related genes (Xnrs) by extracellular Xenopus antivin/lefty (Xatv/Xlefty)-mediated functional antagonism and Brachyury-mediated transcriptional suppression. A strong knockdown of Xlefty/Xatv function was achieved by mixing translation- and splicing-blocking morpholino oligonucleotides that target both the A and B alloalleles of Xatv. Secreted and cell-autonomous inhibitors of Xnr signaling were used to provide evidence that Xnr-mediated induction was inherently long-range in this situation in the large amphibian embryo, essentially being capable of spreading over the entire animal hemisphere. There was a greater expansion of the Organizer and mesendoderm tissues associated with dorsal specification than noted in previous Xatv knockdown experiments in Xenopus, with consequent exogastrulation and long-term maintenance of expanded axial tissues. Xatv deficiency caused a modest animal-ward expansion of the marginal zone expression territory of the Xnr1 and Xnr2 genes. In contrast, introducing inhibitory Xbra-En(R) fusion constructs into Xatv-deficient embryos caused a much larger increase in the level and spatial extent of Xnr expression. However, in both cases (Xatv/Xlefty-deficiency alone, or combined with Xbra interference), Xnr2 expression was constrained to the superficial cell layer, suggesting a fundamental tissue-specific competence in the ability to express Xnrs, an observation with direct implications regarding the induction of endodermal vs. mesodermal fates. Our experiments reveal a two-level suppressive mechanism for restricting the level, range, and duration of Xnr signaling via extracellular inhibition by Xatv/Xlefty coupled with potent indirect transcriptional repression by Xbra.

Inhibins are expressed in the adrenal cortex, but little is known of their binding or role in the adrenal. The aims of the present study were, first, to establish whether a mouse adrenocortical (AC) cell line expresses inhibins/activins and bone morphogenetic proteins (BMP), along with proteins required for inhibin to antagonise activin and BMP actions and, secondly, to characterise and compare inhibin binding sites and proteins in the rat adrenal gland and AC cells. AC cells were found to: (1) express mRNA for multiple BMPs (BMP-2, -3, -4, -6, -8a), growth/differentiation factors (GDF-1, -3, -5, -9), Lefty A and B, and the inhibin alpha, beta(A) and beta(B) subunits (2) secrete inhibin A and inhibin B and (3) express mRNA encoding the inhibin co-receptor, betaglycan, along with activin and BMP type I (ALK2-7) and type II (ActRII, ActRIIB, BMPRII) receptors, and binding proteins (follistatin, BAMBI, gremlin). When applied to sections of rat adrenal glands, [(125)I]inhibin A specifically bound to cells of the adrenal cortex, mainly in the zona reticularis. Scatchard analyses of in vitro [(125)I]inhibin A binding to dispersed rat adrenal cells and AC cells revealed sites of high affinity (K(d)(1) of 0.18 and 0.15 nM, respectively) and low affinity (K(d)(2) of 2.6 and 1.3 nM, respectively. Competition for [(125)I]inhibin A binding by activin A or B (30 nM) was negligible, whereas BMP-2, -6 and -7 competed for between 21 and 33% of specific inhibin A binding (IC(50) between 0.2 and 0.3 nM). Inhibin B crossreaction with inhibin A binding sites was < 8%. Multiple binding protein complexes (molecular weight ranging from 35 to>> 220 kDa) were affinity labelled by [(125)I]inhibin A on both the primary rat adrenal and AC cells. The species of>> 220 kDa were shown by immunoprecipitation to include betaglycan, the species of 105 kDa is consistent in size with type II receptors for activin/BMP, and that of 62 kDa co-migrates with the inhibin-follistatin complex. In summary, the results show that inhibin A binds selectively and with both high and low affinity to AC cells via multiple binding proteins, including a single betaglycan-like species. The results support the role of glycosylated betaglycan in the high affinity binding of inhibin A, but provide consistent evidence from two independent sources of adrenal cells that inhibin A interacts with several membrane proteins in addition to those currently understood to mediate the anti-activin/BMP actions of inhibin.

OBJECTIVE

• To compare the expressions of common fibrosis-relevant genes in hydronephrosis-induced fibrotic renal tissues and normal human renal tissues, thereby providing insights into the cellular and molecular mechanisms of renal fibrosis resulting from hydronephrosis.

METHODS

• A total of 12 extensively fibrotic renal tissue samples from patients with hydronephrosis (H-group) and six normal renal tissue samples from patients who underwent nephrectomy for renal cell carcinoma (N-group), along with their clinical data, were collected at Renmin Hospital of Wuhan University in China between October 2005 and August 2007. • These tissue samples were compared for their transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) pathway-related gene profiles using a real-time polymerase chain reaction (PCR) microarray. • Subsequently, reverse transcriptase-PCR assays were used to validate the expression changes of left-right determination factor (LEFTY), a gene of interest, at the mRNA level. • The different expression of LEFTY at the protein level was confirmed by western blotting and immunohistochemistry assays.

RESULTS

• The results showed that 49 genes were differently expressed in fibrotic renal tissues relative to normal control tissues. Among these genes, 25 were up-regulated and 24 were down-regulated. • LEFTY-B, one of the most markedly altered genes, was down-regulated 13.55-fold compared with N-group tissues. • RT-PCR showed that the LEFTY-A (6.05-fold down-regulated, P < 0.001) and LEFTY-B (12.5-fold down-regulated, P < 0.001) genes, two members of the LEFTY family in human tissues, were both significantly down-regulated in H-group tissues. • Similarly, down-regulations of LEFTY-A (0.25-fold vs N-group, P < 0.001) and LEFTY-B (0.20-fold vs N-group, P < 0.001) proteins were detected by western blotting (P < 0.001). • Immunohistochemical staining showed different distributions of LEFTY in the two tissue samples, and quantitative image analyses confirmed that LEFTY protein expression was lower in H-group tissues than in N-group tissues (P < 0.001).

CONCLUSIONS

• The gene and protein expressions of LEFTY were found to be down-regulated in extensively fibrotic renal tissues induced by hydronephrosis. • LEFTY may represent an ideal candidate for a therapeutic target for renal fibrosis.

Human endometrium is a unique tissue that undergoes sequential phases of proliferation, and secretory changes followed by tissue shedding and bleeding during menstruation. Tissue remodeling is a distinct feature of human endometrium in the secretory phase which prepares endometrium for implantation during the "receptive phase" of the cycle. A discrete dissolution of extracellular matrix (ECM) by a host of enzymes called matrix metalloproteases (MMP) is required for a successful implantation. In the absence of implantation, as a result of progesterone withdrawal, human endometrium loses its receptive state in the premenstrual period and subsequently undergoes a generalized breakdown of ECM by MMPs during menstruation. The homeostasis of ECM of endometrium and the delicate balance between its synthesis and degradation appear to be mediated by reciprocal interaction between TGF-beta and ebaf (lefty) signaling. While TGF-beta acts as a pro-fibrogenic cytokine and maintains the integrity of ECM in endometrium, expression of lefty is associated with events that lead to destruction of ECM facilitating tissue shedding.