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.

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.

Introduction of a normal human chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression.

The development of Wilms' tumor, a pediatric nephroblastoma, has been associated with a deletion in the p13 region of chromosome 11. The structure and function or functions of this deleted genetic material are unknown. The role of this deletion in the process of malignant transformation was investigated by introducing a normal human chromosome 11 into a Wilms' tumor cell line by means of the microcell transfer technique. These variant cells, derived by microcell hybridization, expressed similar transformed traits in culture as the parental cell line. Furthermore, expression of several proto-oncogenes by the parental cells was unaffected by the introduction of this chromosome. However, the ability of these cells to form tumors in nude mice was completely suppressed. Transfer of other chromosomes, namely X and 13, had no effect on the tumorigenicity of the Wilms' tumor cells. These studies provide support for the existence of genetic information on chromosome 11 which can control the malignant expression of Wilms' tumor cells.

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.

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.

Characterization of the mouse transforming growth factor-beta 1 promoter and activation by the Ha-ras oncogene.

We have cloned and sequenced a mouse genomic transforming growth factor beta 1 (TGF-beta 1) DNA fragment that includes the 5' untranslated and regulatory regions of the gene. High-sequence homology with the human TGF-beta 1 gene (66% nucleotide identity in 2.7 kb of DNA upstream of the translational start site) suggested evolutionary conservation of transcriptional regulation for TGF-beta 1. The absence of TATA or CAAT box sequences but the presence of several Sp1-binding and AP-2-like sequences in the promoter region was noted, as previously reported for the human gene. Two transcriptional initiation sites separated by 290 bp were identified by S1 nuclease analysis; these corresponded to transcripts with 866 and 576 nucleotides of 5' untranslated leader sequence. S1 analysis of different mouse tissues indicated that the two transcripts were present in the same ratio even though the total level of TGF-beta 1 mRNA transcripts varied between tissues. Promoter activity adjacent to both transcriptional start sites was demonstrated by using chloramphenicol acetyltransferase fusion genes assayed in mouse AKR-2B fibroblast cells. Transcriptional activation of the promoter by the Ha-ras oncogene was also demonstrated. The minimal promoter constructs (113 and 104 bp 5' of the first and second transcriptional start sites, respectively) were sufficient for induction by Ha-ras. These studies characterize the 5' structure and basal promoter activity of the mouse TGF-beta 1 gene as well as the transcriptional activation of TGF-beta 1 by the Ha-ras oncogene.

Overexpression of transforming growth factor-beta in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene.

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.

Suppression of tumorigenicity in human cell hybrids derived from cell lines expressing different activated ras oncogenes.

Four different human tissue-derived cell lines, each previously shown to express either a Ha-, Ki-, or N-ras-activated oncogene, were fused in four different paired combinations. The three combinations that involved the tumor line HT1080 (activated N-ras oncogene) were found to be tumorigenic in nude mice, but to different degrees. However, the fusion of the tumor lines EJ and SW480 (activated Ha-ras and Ki-ras, respectively) resulted in hybrid cells suppressed for tumorigenicity. The EJ x SW480 hybrids were found to harbor and express both of the activated ras oncogenes. The results suggest that tumorigenic suppression can occur in the presence of two transforming oncogenes of the ras family and that tumorigenicity associated with ras oncogene activation involves additional mechanisms that may differ among tumor cells.

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.

Identification and characterization of the chicken transforming growth factor-beta 3 promoter.

The promoter regions of the three mammalian transforming growth factor-beta genes (TGF-beta s 1, 2, and 3) have been recently cloned and characterized. The sequences show little similarity, suggesting different mechanisms of transcriptional control of these genes. To study differences in transcriptional regulation of mammalian and avian TGF-beta, we have cloned and sequenced the 5'-flanking region of chicken TGF-beta 3. Characterization of this region showed a TATA box and cAMP-responsive element (CRE) and AP-2 binding site consensus sequences starting at 12 and 28 base pairs, respectively, upstream from the TATA box. Moreover, four additional AP-2-like sites, 10 binding sites for the transcription factor Sp1, as well as two AP-1-like sites were also identified. Except for 32 base pairs of identity centered around the TATA box and CRE site and four other relatively small regions of identity, the chicken TGF-beta 3 promoter was found to be structurally very different from the human TGF-beta 3 promoter. Promoter fragments were cloned into a chloramphenicol acetyltransferase reporter plasmid to study functional activity. Basal transcriptional activity of the promoter was regulated in quail fibrosarcoma QM7 cells and in human adenocarcinoma A375 cells by multiple upstream elements including the TATA, CRE, and AP-2 sites. As in the human TGF-beta 3 promoter, the CRE site showed activation by forskolin, an effect which could be shown by expression of TGF-beta 3 mRNA in cultured chicken and quail cells as well. Our results indicate a complex pattern of transcriptional regulation of the chicken TGF-beta 3 gene and suggest that differences in the regulation of expression of the genes for mammalian and avian TGF-beta 3 may result in part from the unique structure of their 5'-flanking regions.

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.

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.

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.

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.

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.

Suppression of tumorigenicity with continued expression of the c-Ha-ras oncogene in EJ bladder carcinoma-human fibroblast hybrid cells.

A human tumor cell line (EJ) expressing an activated c-Ha-ras oncogene was fused with a normal human fibroblast cell line. This fusion resulted in hybrids that behaved as transformed cells in culture but failed to form tumors in nude (athymic) mice. After repeated cell passage, two tumorigenic segregants of the hybrids arose in culture. The levels of expression of activated c-Ha-ras mRNA and its protein product, p21, were similar in the EJ cell line, the nontumorigenic hybrids, and the tumorigenic segregants. DNA transfections of the hybrids were performed with activated c-Ha-ras plasmid constructs, and transfectants expressing a 2-fold level of c-Ha-ras relative to the hybrid cells were found to maintain the nontumorigenic phenotype. We suggest that expression of the active c-Ha-ras oncogene is insufficient for the malignant transformation of these human cells.

Inhibition of growth by transforming growth factor-beta following fusion of two nonresponsive human carcinoma cell lines. Implication of the type II receptor in growth inhibitory responses.

Loss of growth regulation by transforming growth factor-beta (TGF-beta) may be an important step in carcinogenesis. We have used a cell fusion system to show that inhibition of growth by TGF-beta can be restored to carcinoma cell lines that are unresponsive to the inhibitory effects of TGF-beta. In a previous study, the EJ bladder carcinoma line was fused to the SW480 colon adenocarcinoma line and found to produce nontumorigenic hybrid cells along with one hybrid cell clone of low tumorigenicity. Here we show that the capacity of the nontumorigenic hybrid cells to respond to either TGF-beta 1 or TGF-beta 2 has been restored, while the parental or tumorigenic hybrid cells show little or no inhibition of growth following TGF-beta treatment. Cross-linking analyses with labeled TGF-beta 1 demonstrated much higher levels of the type II (85 kDa) receptor in the hybrid cells compared with the parental tumor lines. Both the parental and tumorigenic hybrid cell lines were capable of responding to TGF-beta as evidenced by increased levels of mRNA for fibronectin, type IV collagenase, and plasminogen activator inhibitor after treatment with TGF-beta 1. These results suggest that the type II receptor is necessary for mediating the effects of TGF-beta on inhibition of growth but not on gene activation of the hybrid cells.

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.