Impact of the heavy-quark matching scales in PDF fits.

We investigate the impact of displaced heavy-quark matching scales in a global fit. The heavy-quark matching scale µ m determines at which energy scale µ the QCD theory transitions from N F to N F + 1 in the variable flavor number scheme (VFNS) for the evolution of the parton distribution functions (PDFs) and strong coupling α S ( µ ) . We study the variation of the matching scales, and their impact on a global PDF fit of the combined HERA data. As the choice of the matching scale µ m effectively is a choice of scheme, this represents a theoretical uncertainty; ideally, we would like to see minimal dependence on this parameter. For the transition across the charm quark (from N F = 3 to 4), we find a large µ m = µ c dependence of the global fit χ 2 at NLO, but this is significantly reduced at NNLO. For the transition across the bottom quark (from N F = 4 to 5), we have a reduced µ m = µ b dependence of the χ 2 at both NLO and NNLO as compared to the charm. This feature is now implemented in xFitter 2.0.0, an open source QCD fit framework.

PINP: a serum biomarker of bone formation in the rat.

Serum PINP has emerged as a reliable marker of bone turnover in humans and is routinely used to monitor bone formation. However, the effects of PTH (1-34) on bone turnover have not been evaluated following short-term treatment. We present data demonstrating that PINP is an early serum biomarker in the rat for assessing bone anabolic activity in response to treatment with PTH (1-38). Rat serum PINP levels were found to increase following as few as 6 days of treatment with PTH (1-38) and these increases paralleled expression of genes associated with bone formation, as well as, later increases in BMD. Additionally, PINP levels were unaffected by treatment with an antiresorptive bisphosphonate. PINP may be used to detect PTH-induced early bone formation in the rat and may be more generally applicable for preclinical testing of potential bone anabolic drugs.

Differential effects of estrogen and raloxifene on messenger RNA and matrix metalloproteinase 2 activity in the rat uterus.

A detailed analysis of the differential effects of estrogen (E) compared to raloxifene (Ral), a selective estrogen receptor modulator (SERM), following estrogen receptor (ER) binding in gynecological tissues was conducted using gene microarrays, Northern blot analysis, and matrix metalloproteinase (MMP) 2 activity studies. We profiled gene expression in the uterus following acute (1 day) and prolonged daily (5 wk) treatment of E and Ral in ovariectomized rats. Estrogen regulated twice as many genes as Ral, largely those associated with catalysis and metabolism, whereas Ral induced genes associated with cell death and negative cell regulation. Follow-up studies confirmed that genes associated with matrix integrity were differentially regulated by Ral and E at various time points in uterine and vaginal tissues. Additional experiments were conducted to determine the levels of MMP2 activity in uterus explants from ovariectomized rats following 2 wk of treatment with E, Ral, or one of two additional SERMs: lasofoxifene, and levormeloxifene. Both E and lasofoxifene stimulated uterine MMP2 activity to a level twofold that of Ral, whereas levormeloxifene elevated MMP2 activity to a level 12-fold that of Ral. These data show that one of the significant differences between E and Ral signaling in the uterus is the regulation of genes and proteins associated with matrix integrity. This may be a potential key difference between the action of SERMs in the uterus of postmenopausal women.

Bone anabolic effects of sonic/indian hedgehog are mediated by bmp-2/4-dependent pathways in the neonatal rat metatarsal model.

A neonatal rat metatarsal organ culture model has been employed to study the anabolic effects of Sonic/Indian hedgehog and BMP-4. In this culture system, a significant increase in endochondral ossification is measured by an increase in length of mineralized bone, after daily treatment for 7 days with Sonic hedgehog protein (Shh-N). Previous evidence indicated that PTH related protein (PTHrP) is a critical target of hedgehog function in endochondral ossification. Using a PTHrP blocking antibody, it is shown that hedgehog mediates this activity via pathways other than through PTHrP. A dose-related increase in endochondral ossification is observed when metatarsals are treated with 25 ng/ml recombinant human bone morphogenetic protein 4 (BMP-4). However, this activity is not evident at higher doses of BMP-4 (200 ng/ml). High doses of BMP-4 resulted in increased expression of noggin messenger RNA and cotreatment of noggin and Shh-N resulted in reversal of the anabolic action of Shh-N. This observation suggests that BMP-4 signaling can influence the Shh-N mediated increase in endochondral ossification. Finally, we show that the Shh-N and BMP-4 mediated increase in endochondral ossification is reversed by treatment with antisense oligonucleotides targeted against Cbfa1. Thus, this report identifies Shh-N as an inducer of endochondral ossification that mediates its effect via BMP-4 and Cbfa1-dependent pathways.

Regulation of the promoters for the human bone morphogenetic protein 2 and 4 genes.

The bone morphogenetic proteins 2 and 4 are known to be important in bone formation and are expressed in both the developing and adult mammalian bone. Understanding the regulation of these genes in osteoblasts may yield methods by which we can control expression to induce bone formation. We have isolated and characterized the human BMP-2 and BMP-4 promoters and report substantially more upstream sequence information than that which has been published. Human osteoblasts were found to have a single transcript initiation site that is conserved across species, rather than multiple start sites, as has previously been reported (Feng, J.Q., Harris, M.A., Ghosh-Choudhury, N., Feng, M., Mundy, G.R., Harris, S.E., 1994. Structure and sequence of mouse morphogenetic protein-2 gene (BMP-2): comparison of the structures and promoter regions of BMP-2 and BMP-4 genes. Biochim. Biophys. Acta 1218, 221-224; Heller, L.C., Li, Y., Abrams, K.L., Rogers, M.B., 1999. Transcriptional regulation of the Bmp2 gene. J. Biol. Chem. 274, 1394-1400; Sugiura, T., 1999. Cloning and functional characterization of the 5'-flanking region of the human bone morphogenetic protein-2 gene. Biochem. J. 338, 433-440). A series of promoter deletions for both human BMP-2 and BMP-4 fused to the luciferase reporter gene were analyzed thoroughly in human and murine osteoblastic cell lines. Several compounds and growth factors that stimulate general or osteogenic pathways were used to treat cells transfected with the promoter constructs. Retinoic acid compounds and the phorbol ester, PMA were found to stimulate BMP-2 and, to a lesser degree, BMP-4. The combination of all trans-RA and PMA caused a synergistic increase in BMP-2 promoter activity and endogenous mRNA. The RA stimulation appears to be an indirect effect on the BMP-2 promoter, as the most highly conserved RRE in the BMP-2 promoter was unable to functionally bind or compete for protein binding. Potential binding sites in both promoters for the bone-specific transcription factor, Cbfa-1, were found to specifically bind Cbfa-1 protein in osteoblast nuclear extracts; however, deletion of these sites did not significantly affect transcriptional activity of the promoters in osteoblasts. These data thus present new sequence and regulatory information for the human BMP-2 and BMP-4 promoters and clarify the human BMP-2 gene transcriptional start site in osteoblasts.

Decreased bone mass and bone elasticity in mice lacking the transforming growth factor-beta1 gene.

Transforming growth factor-beta1 (TGF-beta1) knockout (TGF-beta1(-/-)) mice were used to investigate the role of TGF-beta1 in postnatal bone development. Volumetric bone mineral density (BMD) and mineral content (BMC) in these mice and in their normal (TGF-beta1(+/+)) and heterozygous (TGF-beta1(+/-)) littermates were analyzed by quantitative computed tomography (pQCT). Analysis of the proximal tibial metaphysis showed a significant decrease in the BMC of the TGF-beta1(-/-) mice compared to TGF-beta1(+/+) or TGF-beta1(+/-) mice; however, no significant difference was observed in BMD between the groups of mice. pQCT analysis of the tibial midshaft diaphysis showed no difference in the BMD or BMC of cortical bone between the groups. Histomorphometry revealed no significant difference in trabecular connectivity or in trabecular bone volume, number, or thickness. However, the width of the tibial growth plate and the longitudinal growth rate were significantly decreased in the TGF-beta1(-/-) mice, resulting in shorter tibia. Acoustic velocity measurements showed significant differences between the groups of mice with an apparent dosage effect of TGF-beta1 expression on the anisotropic properties of the bone. These data show that longitudinal growth and total mineral content are affected in mice lacking TGF-beta1, as well as the elastic properties of the bone, consistent with an important role for TGF-beta1 in bone modeling and bone quality.

Up-regulation of cytokine mRNA, adhesion molecule proteins, and MHC class II proteins in salivary glands of TGF-beta1 knockout mice: MHC class II is a factor in the pathogenesis of TGF-beta1 knockout mice.

Mice homozygous for a disrupted TGF-beta1 allele develop multiple lymphoproliferative disorders similar to those seen in the pseudolymphoma of Sjögren's syndrome. At 2 wk of age, these TGF-beta1 mutant mice begin to develop wasting syndrome and die at around 4 to 5 wk of age. We studied salivary glands from symptomatic mutant mice >14 days of age. Reverse transcriptase-PCR analysis showed up-regulation of proinflammatory cytokine genes such as IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, and IFN-gamma in these mutant mice. Enhanced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and MHC class II as well as CD4-positive T lymphocyte infiltration was detected by immunostaining. To elucidate the role of MHC class II, salivary glands from TGF-beta1/MHC class II double knockout mice were used to investigate the expression of adhesion molecules and MHC class II. In spite of the existence of basal intercellular adhesion molecule-1 expression on vessels, there was neither MHC class II expression, enhanced vascular cell adhesion molecule-1 expression, nor lymphocytic infiltration in the salivary glands. These results suggest that MHC class II plays a significant role in the pathogenesis of TGF-beta1 mutant mice. Although the mechanism that initiates multiple inflammatory diseases in these mice remains unclear, the context reported here would provide insight into the immunopathology of Sjögren's syndrome.

Autoimmunity associated with TGF-beta1-deficiency in mice is dependent on MHC class II antigen expression.

The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

The liver in transforming growth factor-Beta-1 (TGF-beta 1) null mutant mice.

Transforming growth factor-beta-1 (TGF-beta 1) null mutant mice have no gross developmental abnormalities at birth but succumb to multifocal inflammatory lesions that lead to organ failure and death about 20 days after birth. Treatment with anti-inflammatory and immune suppressive agents, such as rapamycin, reduces the severity and extent of inflammatory infiltrates in the liver and can prolong the life of knockout (KO) mice compared to untreated null mice. To determine whether there is an associated hepatic phenotype, livers of "young" (< 3 weeks), "old" (> 3 weeks), and age-matched wild-type (WT) mice were studied using light and electronmicroscopy. On light microscopy, old KO mice had foci of mononuclear cells in liver parenchyma in addition to scattered foci of megalocytosis. Intracytoplasmic vacuoles, some of which were juxtanuclear in location, were also seen but these were most prominent in the oldest (10 weeks) rapamycin-treated mouse. In the untreated young KO mice, there were only foci of mononuclear cells in the liver parenchyma and portal tracts and variable numbers of binucleated hepatocytes. Ultrastructurally, there was a significant increase in the number of mitochondria in livers of the old KO mice, when compared either to the age-matched wild-type or to the young KO mice (p > .001). Hepatocytes from all KO mice showed increased numbers of hypertrophied or enlarged Golgi complexes compared to age-matched wild-type mice. Intracytoplasmic canaliculi lined with microvilli were seen in livers of old KO mice, but were absent in the young KO and wild-type mice. Primary cultures of hepatocytes, derived from livers of both young and old KO mice, showed similar changes on phase contrast and electronmicroscopy. These included juxtanuclear vacuoles, intracytoplasmic canaliculi, enlarged Golgi vesicles, and increased numbers of autolysosomes. Phenotypic abnormalities of mitochondria were either minimal or absent in cultured KO hepatocytes. The findings demonstrate, for the first time, that targeted disruption of the TGF-beta 1 gene in mice results in an altered ultrastructural phenotype of hepatocytes. The data suggest that TGF-beta 1 may be required for normal development and regulation of subcellular organelles in hepatocytes and may be essential for physiological functions involving mitochondria and Golgi complex.

Control of nitric oxide production by endogenous TGF-beta1 and systemic nitric oxide in retinal pigment epithelial cells and peritoneal macrophages.

Both in vivo and in vitro experiments demonstrate that transforming growth factor-beta1 (TGF-beta1) suppresses expression of the inducible form of nitric oxide synthase (iNOS). In this study, we examined the effects of exogenous and endogenous TGF-beta1 on retinal pigment epithelial (RPE) cells and resident peritoneal macrophages ex vivo using cells from TGF-beta1 null (TGF-beta1-/-) mice or age-matched wild-type (TGF-beta1+/+) or heterozygous (TGF-beta1+/-) littermates. RPE cells from both TGF-beta1-/- mice and TGF-beta1+/+ littermates produced NO and were immunocytochemically positive for iNOS protein only following treatment with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS); however, RPE cells from TGF-beta1-/- mice produced 40% more NO than cells from TGF-beta1+/+ mice. In contrast, resident peritoneal macrophages from both TGF-beta1+/+ and TGF-beta1-/- mice expressed iNOS protein without stimulation and in the absence of detectable production of NO. The expression of iNOS was increased by treatment with IFN-gamma, resulting in detectable levels of NO. Macrophages from TGF-beta1+/+ mice appeared to produce NO in a manner inversely proportional to the serum content of NO2- and NO3- of the mice from which the cells were obtained; no such correlation existed in TGF-beta1+/- or TGF-beta1-/- mice. Treatment of RPE cells or macrophages from both TGF-beta1+/+ and TGF-beta1-/- mice with exogenous TGF-beta1 decreased both iNOS protein and NO production. These findings demonstrate a novel role of endogenous TGF-beta1 in coupling systemic NO production to the production of NO by macrophages, and demonstrate that endogenous and exogenous TGF-beta1 can act differently to suppress NO production.

Spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta 1 null mouse.

Transforming growth factor beta 1 null mice (TGF-beta 1-/-) suffer from multifocal inflammation and die by 3-4 wk of age. In these mice, levels of nitric oxide (NO) reaction products in serum are elevated approximately fourfold over levels in controls, peaking at 15-17 d of life. Shortterm treatment of TGF-beta 1-/- mice with NG-monomethyl-L-arginine suppressed this elevated production of NO. Expression of inducible NO synthase (iNOS) mRNA and protein is increased in the kidney and heart of TGF-beta 1-/- mice. These findings demonstrate that TGF-beta 1 negatively regulates iNOS expression in vivo, as had been inferred from mechanistic studies on the control of iNOS expression by TGF-beta 1 in vitro.

Characterization of the promoter region of the human transforming growth factor-beta type II receptor gene.

Diminished cellular responsiveness to transforming growth factor-beta (TGF-beta) is frequently correlated with decreased transcription of the type II receptor for TGF-beta (TGF-beta RII). We have cloned and characterized the human TGF-beta RII promoter and, using S1 nuclease mapping and 5' rapid amplification of cDNA ends polymerase chain reaction, have identified five alternative transcription start sites within the region -33 to +57. DNA transfection experiments and electrophoretic mobility shift assays have revealed the existence of five distinct regulatory regions including two positive regulatory elements and two negative regulatory elements in addition to the core promoter region. The first positive regulatory element (-219 to -172) interacts with two distinct nuclear protein complexes, at least one of which appears to be a previously unidentified transcription factor. The second positive regulatory element (+1 to +35) also interacts with two separate protein complexes, both of which appear to be novel transcription factors. Deletion of either positive regulatory element markedly decreased expression of the target gene, suggesting that both positive regulatory elements are necessary for basal expression levels of TGF-beta RII.

SLE-like autoantibodies and Sjögren's syndrome-like lymphoproliferation in TGF-beta knockout mice.

Mice bearing the TGF-beta 1 null mutation (-/-) develop lymphoid infiltrates in the heart, lungs, salivary glands, and other organs similar to those seen in the pseudolymphoma of Sjögren's Syndrome. We studied sera from -/- mice and found elevated Ab levels to dsDNA, ssDNA, and Sm ribonucleoprotein. No Abs to SSA/Ro or SSB/La and no IgM rheumatoid factor were found. Serum autoantibodies were predominately IgG and were specific as shown by ELISA inhibition studies. Antinuclear Ab patterns on Western blots varied from one mouse to the next, indicating a random process responsible for the diversity. Wild-type and heterozygote mice had no autoantibodies. Ig glomerular deposits were found in -/- mice, indicating that these autoantibodies may be pathogenic. Treatment of -/- mice with dexamethasone or TGF-beta 1 failed to suppress autoantibody production. These mice represent an overlap combining the autoimmune serology of SLE with the tissue infiltrates of SS. Our results support the concept that TGF-beta 1 is an important naturally occurring immunosuppressive cytokine whose absence can lead to a systemic autoimmune disease.

Maternal rescue of transforming growth factor-beta 1 null mice.

Maternal sources of transforming growth factor-beta 1 (TGF-beta 1) are shown here to contribute to the normal appearance and perinatal survival of TGF-beta 1 null newborn mice. Labeled TGF-beta 1 crossed the placenta and was recovered intact from various tissues after oral administration to mouse pups. TGF beta-1 protein was also detected in cells recovered from breast milk. In immunohistochemical analyses, TGF-beta 1 null embryos and null newborn pups born to TGF-beta 1 heterozygotes stained positive for TGF-beta 1, whereas those born to a null female were negative and had severe cardiac abnormalities. These results suggest an important role for maternal sources of TGF-beta 1 during development and, more generally, provide evidence for maternal rescue of targeted gene disruption in the fetus.

Transforming growth factor beta 1 (TGF-beta 1) controls expression of major histocompatibility genes in the postnatal mouse: aberrant histocompatibility antigen expression in the pathogenesis of the TGF-beta 1 null mouse phenotype.

The phenotype of the transforming growth factor beta 1 (TGF-beta 1) null mouse has been previously described and is characterized by inflammatory infiltrates in multiple organs leading to a wasting syndrome and death as early as 3 weeks after birth. Since this phenotype occurs in the absence of any detectable pathogen, potential autoimmune disease mechanisms were investigated. We examined major histocompatibility complex (MHC) mRNA expression in tissues of the TGF-beta 1 null mouse and found levels of both the class I and class II MHC mRNA elevated compared to normal or TGF-beta 1 heterozygous littermates. This elevated expression was seen prior to any evidence of inflammatory infiltrates, suggesting a causal relationship between increased MHC expression and activation of immune cell populations. Cell surface expression of MHC molecules was detected by immunohistochemistry and correlated well with mRNA levels. Expression of mRNA for interferon gamma and its receptor was unchanged at the ages when increased MHC expression became apparent. Down-regulation of class I MHC expression by TGF-beta 1 was also demonstrated in vitro in fibroblasts isolated from TGF-beta 1 null mice. These findings suggest that one natural function of TGF-beta 1 is to control expression of both MHC classes. Altered regulation of MHC expression may be a critical step leading to the multifocal inflammation and wasting syndrome seen in the TGF-beta 1 null mouse. These results suggest potential applications for TGF-beta in the management of autoimmune disease, allograft rejection, and other problems associated with altered MHC expression.

Synergistic increase of phorbol ester-induced c-fos mRNA expression by retinoic acid through stabilization of the c-fos message.

Retinoic acid (RA) has been shown to be able to antagonize or synergize with phorbol 12-myristate 13-acetate (PMA). In contrast to its antagonistic effects on PMA-dependent gene expression, no molecular target or mechanism of synergism has been characterized yet. We now report, that RA synergistically enhances the induction of c-fos, but not c-jun mRNA by PMA in cells whose growth was stimulated by RA alone. The responding cells were hybrids of tumor cell lines whose growth and PMA-dependent c-fos mRNA expression remained unaffected by RA. The increase in PMA-dependent c-fos expression required pretreatment of cells with RA for at least 2-4 h and was achieved at doses as low as 10(-10) M. Nuclear run-on experiments and transient transfection assays using a chimeric reporter gene construct with sequences from the c-fos promoter indicated that RA did not affect PMA-dependent c-fos transcription. Instead, RA stabilized the c-fos message after induction by PMA as assessed by measuring the half-life of c-fos mRNA in actinomycin D-treated cells. This post-transcriptional regulation provides a mechanism whereby RA can synergistically enhance gene expression by PMA.

Regulation of the transforming growth factor-beta 1 and -beta 3 promoters by transcription factor Sp1.

The promoter regions of the genes encoding the three mammalian transforming growth factors-beta (TGF-beta 1, -beta 2, and -beta 3) show little similarity in sequence, suggesting diverse transcriptional control. As a step towards understanding transcriptional regulation of the individual TGF-beta genes we tested each of the three TGF-beta promoter regions (pTGF-beta) for stimulation by the transcription factor Sp1, given that several possible Sp1-binding sites were identified by sequence analysis in pTGF-beta 1 and pTGF-beta 3. A Drosophila melanogaster cell culture system was employed to examine expression levels of pTGF-beta::cat constructs coexpressed with an Sp1 expression plasmid in a cell background devoid of any Sp1 homolog. While both pTGF-beta 1 and pTGF-beta 3 were strongly stimulated by Sp1, pTGF-beta 2 was completely unaffected. Promoter fragments of the TGF-beta 1 and TGF-beta 3 genes, but not TGF-beta 2 were able to compete for binding of Sp1 to DNA oligomers containing consensus Sp1-binding sites. Moreover, specific binding to pTGF-beta 1 and pTGF-beta 3 fragments was seen using pure Sp1 or nuclear protein extracts. Thus, TGF-beta 1 and TGF-beta 3 (but not TGF-beta 2) are regulated by the transcription factor Sp1, indicating differential transcriptional regulation of genes whose protein products are functionally very similar.

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland.