Mammary epithelial cell phagocytosis downstream of TGF-ß3 is characterized by adherens junction reorganization.

After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-ß (TGF-ß) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-ß enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-ß3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and ß-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or ß-catenin reduced the effect of TGF-ß3 on phagocytosis to near baseline levels. However, ß-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and ß-catenin signaling contribute to phagocytosis downstream of TGF-ß3. Our data provide insight into how mammary epithelial cells contribute to apoptotic cell clearance, and in light of the negative consequences of impaired apoptotic cell clearance during involution, may shed light on involution-associated breast pathologies.

Smad3 loss confers resistance to the development of trinitrobenzene sulfonic acid-induced colorectal fibrosis.

BACKGROUND: Transforming growth factor-beta (TGF-beta)/Smad3 signalling plays a central role in tissue fibrogenesis, acting as a potent stimulus of extracellular matrix (ECM) protein accumulation. The aim of this study was to evaluate the potential role of Smad3 in the pathogenesis of colonic fibrosis induced by trinitrobenzene sulfonic acid (TNBS) in Smad3 null mice. MATERIALS AND METHODS: Chronic colitis-associated fibrosis was induced in 15 Smad3 null and 13 wild-type mice by intra-rectal administration of TNBS. Each mouse received an incremental dose of TNBS (0.5-1.0 mg per week) over a 6-week period. The colon was excised for macroscopic examination and histological, morphometric and immunohistochemical analyses. For immunohistochemistry, alpha-smooth muscle actin (alpha-SMA), collagen types I-III, TGF-beta1, connective tissue growth factor (CTGF), Smad3, Smad7, and CD3 antibodies were used. RESULTS: At macroscopic examination, the colon of Smad3 wild-type mice appeared significantly harder, thicker and shorter than that of the Smad3 null mice. Of the wild-type mice, 50% presented colonic adhesions and strictures. Histological and morphometric evaluation revealed a significantly higher degree of colonic fibrosis and accumulation of collagen in the Smad3 wild-type compared to null mice, whereas the degree of colonic inflammation did not differ between the two groups of mice. Immunohistochemical evaluation showed a marked increase in CTGF, collagen I-III, TGF-beta and Smad3 staining in the colon of Smad3 wild-type compared to null mice, whereas Smad7 was increased only in null mice. CONCLUSIONS: These results indicate that Smad3 loss confers resistance to the development of TNBS-induced colonic fibrosis. The reduced fibrotic response appears to be due to a reduction in fibrogenic mesenchymal cell activation and ECM production and accumulation. Smad3 could be a novel target for potential treatment of intestinal fibrosis, especially in inflammatory bowel disease.

SNIP1 inhibits NF-kappa B signaling by competing for its binding to the C/H1 domain of CBP/p300 transcriptional co-activators.

SNIP1 is a 396-amino acid nuclear protein shown to be an inhibitor of the TGF-beta signal transduction pathway and to be important in suppressing transcriptional activation dependent on the co-activators CBP and p300. In this report we show that SNIP1 potently inhibits the activity of NF-kappa B, which binds the C/H1 domain of CBP/p300, but does not interfere with the activity of transcription factors such as p53, which bind to other domains of p300, or factors such as VP16, which are independent of these co-activators. Inhibition of NF-kappa B activity is a function of the N-terminal domain of SNIP1 and involves competition of SNIP1 and the NF-kappa B subunit, RelA/p65, for binding to p300, similar to the mechanism of inhibition of Smad signaling by SNIP1. Immunohistochemical staining shows that expression of SNIP1 is strictly regulated in development and that it colocalizes, in certain tissues, with nuclear staining for RelA/p65 and for p300, suggesting that they may regulate NF-kappa B activity in vivo in a spatially and temporally controlled manner. These data led us to suggest that SNIP1 may be an inhibitor of multiple transcriptional pathways that require the C/H1 domain of CBP/p300.

Is Smad3 a major player in signal transduction pathways leading to fibrogenesis?

Transforming growth factor (TGF)-beta plays a central role in fibrosis, contributing both to the influx and activation of inflammatory cells, as well as to activation of fibroblasts to elaborate extracellular matrix. In the past few years, new insight has been gained into signal transduction pathways downstream of the TGF-beta receptor serine-threonine kinases with the identification of a family of evolutionarily conserved Smad proteins. Two receptor-activated Smad proteins, Smad2 and Smad3, are phosphorylated by the activated TGF-beta type I receptor kinase, after which they partner with the common mediator, Smad4, and are translocated to the nucleus to where they participate in transcriptional complexes to control expression of target genes. We have shown in wound healing studies of mice null for Smad3, that loss of this key signaling intermediate interferes with the chemotaxis of inflammatory cells to TGF-beta as well as with their ability to autoinduce TGF-beta. Moreover, studies with mouse embryo fibroblasts null for Smad3 show that TGF-beta-dependent induction of c-Jun and c-Fos, important in induction of collagen as well as in autoinduction of TGF-beta, is mediated by Smad3. Based on these observations, we hypothesize that loss of Smad3 will confer resistance to fibrosis and result in reduced inflammatory cell infiltrates, reduced autoinduction of TGF-beta, important to sustain the process, and reduced elaboration of collagen. Preliminary observations in a model of radiation-induced fibrosis confirm this hypothesis and suggest that inhibitors of Smad3 might have clinical application both to improve wound healing and to reduce fibrosis.

Transforming growth factor-beta1 expression in early biopsy specimen predicts long-term graft function following pediatric renal transplantation.

The main cause of late graft loss or declining long-term graft function is chronic allograft nephropathy (CAN), characterized by progressive interstitial fibrosis. Transforming growth factor (TGF)-beta1 plays a key role in fibrogenesis. We immunohistochemically investigated whether the degree of TGF-beta1 expression in early biopsy specimens routinely obtained from stable allografts at 100 d could predict fibrosis and graft dysfunction in the late phase. Patients were children with grafts from related donors. We immunohistochemically determined intracellular and extracellular expression of TGF-beta1 in the graft using LC antibody (LC) for intracellular TGF-beta1 and CC antibody (CC) for extracellular TGF-beta1. The change in creatinine clearance between 100 d and 3 yr after transplantation (DeltaCcr) was used as an index of long-term graft function. We also used image analysis to calculate the relative area involved by interstitial fibrosis in the trichrome-stained section of graft biopsy specimens at 100 d and 3 yr, designating the change as DeltaFI. DeltaCcr was -4.2+/-9.4 mL/min in subjects with minimal early immunoreactivity for CC and -20.5+/-15.9 mL/min in subjects with strong reactivity (p<0.05). DeltaCcr was -14.5+/-18.6 mL/min in subjects with minimal early immunoreactivity for LC and -11.7+/-12.8 mL/min in those with strong reactivity. DeltaFI in subjects with minimal CC reactivity (1.28+/-4.11%) tended to be lower than that in subjects with strong reactivity (8.45+/-15.47%). Neither fibrosis at 100 d nor DeltaFI differed between subjects with minimal and strong LC reactivity. Thus, strong extracellular TGF-beta1 expression in grafts at 100 d after transplantation is associated with a long-term decline in graft function and tends to be associated with increased graft fibrosis at 3 yr.

Transforming growth factors-beta are not good biomarkers of chemopreventive efficacy in a preclinical breast cancer model system.

Using a carcinogen-initiated rat model of mammary tumorigenesis, we tested the hypothesis that transforming growth factor (TGF)-betas are useful biomarkers of chemopreventive efficacy in the breast. The chemopreventive agents tested were tamoxifen and the retinoids 9-cis-retinoic acid (9cRA) and N-(4-hydroxyphenyl)retinamide (4-HPR), because both antiestrogens and retinoids have previously been shown to upregulate TGF-betas in vitro. Despite demonstrable chemopreventive efficacy in this model, none of these agents, alone or in combination, had any significant impact on the expression of TGF-betas in the mammary ductal epithelium or periductal stroma as determined by immunohistochemistry. These data suggest that TGF-betas are not likely to be useful biomarkers of chemopreventive efficacy in a clinical setting.

Immunohistochemical expression of Smads 1-6 in the 15-day gestation mouse embryo: signaling by BMPs and TGF-betas.

The eight mammalian Smad proteins mediate cellular signaling from members of the transforming growth factor-beta (TGF-beta), bone morphogenetic protein (BMP), and activin families. Smads 1, 5, and 8 transmit signals from BMPs, while Smads 2 and 3 transmit signals from TGF-betas and activin. Smad 4 is a common mediator of both pathways, while Smads 6 and 7 inhibit signaling. Signal transduction involves translocation of Smad complexes to the nucleus and subsequent gene activation. Little is known about the expression of endogenous Smad proteins during development. We identified commercially available Smad antibodies that specifically recognize a unique Smad protein and are suitable for immunohistochemistry. Here we compare the localization of Smads 1, 2, 3, 4, 5, and 6 in tissues of the 15-day gestation mouse embryo. Immunoreactive Smad proteins are seen in many tissues with differences in the localization being dependent upon the cell type. All tissues express Smad 4 and at least one each of the BMP-specific and TGF-beta-specific Smads, while expression of Smad 6 is more restricted. Differences are observed in the nuclear versus cytoplasmic localization among the Smads in different cell types or tissues, suggesting selective activation of Smads during this stage of development.

Characterization of the mouse Smad1 gene and its expression pattern in adult mouse tissues.

Smad1 belongs to a family of receptor-activated proteins which mediate signals from TGF-beta superfamily ligands, including TGF-beta and BMPs. Although much is known about the biochemistry of Smad1 signal transduction, the role of Smad1 in vivo is still unclear. Here we present the first description of the genomic structure of the mouse Smad1 gene and the characterization of its expression pattern in adult mouse tissues by immunohistochemistry. The Smad1 gene contains 7 exons and spans >42 kb of genomic DNA. Its coding region is contained within 6 exons and all introns, except intron 1, follow the GT/AG rule. Immunohistochemical analysis shows that Smad1 is widely expressed in adult mouse tissues, with a varying degree of nuclear localization in different cell types, suggesting a regulated function for this protein. This study assigns all of the exon-intron boundaries of the mouse Smad1 gene and provides the basis for assessing the functional significance of this gene using targeted gene manipulation in the mouse.

Differential effects of transforming growth factor-beta(s) and glial cell line-derived neurotrophic factor on gene expression of presenilin-1 in human post-mitotic neurons and astrocytes.

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity.

The new synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a potent, multifunctional molecule. It induces monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts and enhances the neuronal differentiation of rat PC12 pheochromocytoma cells caused by nerve growth factor. CDDO inhibits proliferation of many human tumor cell lines, including those derived from estrogen receptor-positive and -negative breast carcinomas, myeloid leukemias, and several carcinomas bearing a Smad4 mutation. Furthermore, it suppresses the abilities of various inflammatory cytokines, such as IFN-gamma, interleukin-1, and tumor necrosis factor-alpha, to induce de novo formation of the enzymes inducible nitric oxide synthase (iNos) and inducible cyclooxygenase (COX-2) in mouse peritoneal macrophages, rat brain microglia, and human colon fibroblasts. CDDO will also protect rat brain hippocampal neurons from cell death induced by beta-amyloid. The above activities have been found at concentrations ranging from 10(-6) to 10(-9) M in cell culture, and these results suggest that CDDO needs further study in vivo, for either chemoprevention or chemotherapy of malignancy as well as for neuroprotection.

Effects of transforming growth factor-beta (isoforms 1-3) on amyloid-beta deposition, inflammation, and cell targeting in organotypic hippocampal slice cultures.

The transforming growth factor-beta (TGF-beta) family consists of three isoforms and is part of a larger family of cytokines regulating differentiation, development, and tissue repair. Previous work from our laboratory has shown that TGF-beta1 can increase amyloid-beta protein (Abeta) immunoreactive (Abetair) plaque-like deposits in rat brain. The aim of the current study was to evaluate all three isoforms of TGF-beta for their ability to affect the deposition and neurotoxicity of Abeta in an organotypic, hippocampal slice culture model of Abeta deposition. Slice cultures were treated with Abeta either with or without one of the TGF-beta isoforms. All three isoforms can increase Abeta accumulation (over Abeta treatment alone) within the slice culture, as determined by ELISA. However, there are striking differences in the pattern of Abetair among the three isoforms of TGF-beta. Isoforms 1 and 3 produced a cellular pattern of Abeta staining that colocalizes with GS lectin staining (microglia). TGF-beta2 produces dramatic Abeta staining of pyramidal neurons in layers CA1-CA2. In addition to cellular Abeta staining, plaque-like deposits are increased by all of the TGF-betas. Although no gross toxicity was observed, morphological neurodegenerative changes were seen in the CA1 region when the slices were treated with Abeta plus TGF-beta2. Our results demonstrate important functional differences among the TGF-beta isoforms in their ability to alter the cellular distribution and degradation of Abeta. These changes may be relevant to the pathology of Alzheimer's disease (AD).

Transforming growth factor-beta inhibits apoptosis induced by beta-amyloid peptide fragment 25-35 in cultured neuronal cells.

Previously, we demonstrated that transforming growth factor-beta (TGF-beta) pretreatment protects neuroblastoma cell lines, human hNT neurons, and primary rat embryo hippocampal neurons (REHIPs) from degeneration caused by incubation with beta-amyloid peptide (Abeta). Here we present evidence suggesting that TGF-beta interferes with an apoptotic pathway induced by Abeta. TGF-beta preteatment decreases the amount of DNA laddering seen following Abeta treatment in neuroblastoma cells, while in REHIPs, TGF-beta decreases the number of positive cells detected in situ by Klenow labelling following Abeta treatment. RT-PCR shows that in REHIPs, Abeta decreases mRNA expression of Bcl-2, as well as the ratio of Bcl-xL/Bcl-xS, with little effect on Bax expression. These changes are expected to promote apoptosis. When REHIPs are incubated with TGF-beta before addition of Abeta, the Bcl-xL/Bcl-xS ratio and Bcl-2 levels are increased compared to cells treated with Abeta alone. Again there is little effect on Bax expression. Western blotting and immunohistochemistry experiments also show that TGF-beta maintains increased levels of Bcl-2 and Bcl-xL protein in REHIPs even in the presence of Abeta. This pattern of gene expression should function to decrease apoptosis. Similarly, RT-PCR analysis of mRNA prepared from hNT cells shows that TGF-beta pretreatment before addition of Abeta maintains a higher level of Bcl-2 expression and an increased Bcl-xL/Bcl-xS ratio as compared to cells treated with Abeta alone. In neuronal cell types treated with Abeta, TGF-beta appears to regulate expression of genes in the Bcl-2 family to favor an anti-apoptotic pathway.

Expression of transforming growth factor-beta isoforms in the rat male accessory sex organs and epididymis.

We studied the expression and distribution of transforming growth factor-beta (TGF-beta) isoforms in the rat male accessory sex glands and the epididymis. Our data demonstrate the expression of both TGF-beta1 and -beta3 isoforms in ventral prostate (VP), seminal vesicle (SV), coagulating gland (CG), and epididymis (E) by Northern blot analysis. In addition, there was differential expression of TGF-beta3 in the three regions of epididymis, the corpus region being the highest. Immunostaining data showed intense staining for latent TGF-beta1 in all the male accessory glands. In contrast, no staining using antibodies specific for active TGF-beta1 was observed. No expression of TGF-beta2 was evident either by immunohistochemistry or Northern blot analysis. The presence of mature TGF-beta3 protein was observed in the secretory epithelium of VP, CG, and corpus E. There was no detectable staining of TGF-beta3 in the seminal vesicle and caput and cauda regions of epididymis. These data suggest possible differential regulation of TGF-beta isoform expression in the male reproductive system and predict unique roles for individual TGF-beta isoforms in sperm maturation and maintenance.

Transforming growth factor-betas in neurodegenerative disease.

Transforming growth factors-betas (TGF-betas), a family of multifunctional peptide growth factors, affect cells of the central nervous system (CNS). The three mammalian TGF-beta isoforms, TGF-betas 1, 2 and 3, are expressed in adult human brain. Since neuronal degeneration is a defining feature of CNS degenerative diseases, TGF-beta may be important because it can influence neuronal survival. In vitro TGF-beta promotes survival of rat spinal cord motoneurons and dopaminergic neurons. In addition to direct effects on neuronal survival, TGF-beta treatment of cultured astrocytes induces a reactive phenotype. Thus, TGF-beta may also normalize the extracellular matrix environment in degenerative diseases. The expression of TGF-betas change in response to neuronal injury. TGF-beta 1 expression increases in astrocytes and microglia in animal models of cerebral ischemia, while TGF-beta 2 expression increases in activated astroglial cells in human neurodegenerative diseases. TGF-betas protect neurons from a variety of insults. TGF-beta maintains survival of chick telencephalic neurons made hypoxic by treatment with cyanide and decreases the area of infarction when administered in animal models of cerebral ischemia. In vitro TGF-beta protects neurons from damage induced by treatment with beta-amyloid peptide, FeSO4 (induces production of reactive oxygen species), Ca2+ ionophores, glutamate, glutamate receptor agonists and MPTP (toxic for dopaminergic neurons). TGF-beta maintains mitochondrial potential and Ca2+ homeostasis and inhibits apoptosis in neurons. TGF-beta does not prevent neuronal degeneration in a rat model of Parkinson's disease and has yet to be tested in newly developed transgenic mouse models of Alzheimer's disease. TGF-beta is a potent neuroprotective agent which may affect the pathogenesis of neurodegenerative diseases of the CNS.

TGF-beta receptors-I and -II immunoexpression in Alzheimer's disease: a comparison with aging and progressive supranuclear palsy.

The transforming growth factor-betas (TGF-betas) influence cell survival, and TGF-beta2 shows increased immunoexpression in neurofibrillary tangle-bearing neurons and reactive glia in Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). We compared immunohistochemical expression of TGF-beta type I (RI) and type II (RII) receptors in eight patients with AD, eight controls and three cases of progressive supranuclear palsy. Mild intraneuronal immunoreactivity for the RI receptor was observed in all cases. Intraneuronal TGF-beta RII receptor immunoexpression was more common in all groups, and its frequency did not differ between groups. We observed increased immunoreactivity for both RI and RII subtypes in reactive glia in the AD frontal cortex (RI: U = 0.5, p = 0.002; and RII: U = 9.000, p = 0.006) and parahippocampal gyrus (RI: U = 9.500, p = 0.013; RII: U = 14.5, p = 0.05) compared to control cases. We conclude that TGF-beta RI and II immunoreactivity is increased in reactive glia in AD and progressive supranuclear palsy, and RI immunoreactivity may occasionally be increased in neurons in cases with advanced AD.

Inducible nitric oxide production and expression of transforming growth factor-beta1 in serum and CSF after cerebral ischaemic stroke in man.

A residual blood supply to the ischaemic brain is a crucial determinant for tissue survival. Early changes in the vascular network and subsequent angiogenesis may be mediated by short-lived molecules like nitric oxide (NO) or growth factors such as transforming growth factor-beta1 (TGF-beta1). Although TGF-beta1 can inhibit NO production, this interaction has not been studied after ischaemia in humans. Serum samples were taken from patients at 24 h and 6 months and cerebrospinal fluid (CSF) samples at 24 h and 1 week later for possible correlation between the two factors. Tissue expression of TGF-beta1 and of the inducible isoform of NO synthase (NOS2) was assessed by immunohistochemistry. CSF levels of NO2-/NO3- as well as total (active + latent) TGF-beta1 were higher in stroke patients as compared to controls 24 h after the stroke. Both NO2-/NO3- and TGF-beta1 were lower 6 months after the stroke compared to 24 h. Levels of NO2-/NO3- correlated with levels of TGF-beta1 within the time points (P = 0.041, Kendall correlation coefficient). There was a strong staining for NOS2 in brain tissue sections in neurones, reactive astrocytes, infiltrating white blood cells, and endothelial cells of larger microvessels. TGF-beta1 expression was mainly limited to neurones and reactive astrocytes. These findings suggest that the interaction between TGF-beta1 and NOS2 might be important for angiogenesis after cerebral ischaemia and may indicate that TGF-beta1 is upregulated as a negative feedback response to elevated levels of NO.

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia.

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor beta1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor beta1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

Transforming growth factor-beta protects human hNT cells from degeneration induced by beta-amyloid peptide: involvement of the TGF-beta type II receptor.

Post-mitotic, human neurons (hNT cells) which have a phenotype similar to that of terminally differentiated neurons of the central nervous system were generated by treating the NT2/D1 human teratocarcinoma cell line with retinoic acid. Treatment of both hNT and NT2/D1 cells with 10(-5) M beta-amyloid peptide fragment 25-35 (A beta P) for 24 h resulted in a decrease in cell viability as determined by MTT incorporation and Trypan blue exclusion, and also induced an apoptotic morphology in hNT cells. Pre-treatment of cells for 24 h with 10 ng/ml TGF-beta 1 or 2 before addition of A beta P reduced the apoptotic morphology of hNT cells and increased cell viability in hNT cells, but not in NT2/D1 cells. Results of RT-PCR, immunohistochemistry and analysis of receptor cross-linking of [125I]TGF-beta 1 to the cell membrane, all showed that the TGF-beta type II receptor is expressed by hNT cells, but not NT2/D1 cells. These results suggest that TGF-beta can protect human, terminally differentiated, TGF-beta type II receptor-positive neurons from A beta P toxicity. We propose that the increased expression of TGF-beta in brains of patients with Alzheimer's disease may offer some degree of neuroprotection if neurons also express a functional TGF-beta type II receptor.

Deficient transforming growth factor-beta1 activation and excessive insulin-like growth factor II (IGFII) expression in IGFII receptor-mutant tumors.

The insulin-like growth factor II receptor (IGFIIR) gene has been identified as a coding region target of microsatellite instability in human gastrointestinal (GI) tumors. IGFIIR normally has two growth-suppressive functions: it binds and stimulates the plasmin-mediated cleavage and activation of the latent transforming growth factor-beta1 (LTGF-beta1) complex, and it mediates the internalization and degradation of IGFII ligand, a mitogen. We used an immunohistochemical approach to determine whether IGFIIR mutation affected expression of these proteins in GI tumors. Four highly specific antibodies were used: LC(1-30), which recognizes the active form of TGF-beta1; anti-LTGF-beta1, which detects the LTGF-beta1 precursor protein; anti-IGFIIR; and anti-IGFII ligand. Twenty GI tumors either with (6 of 20) or without (14 of 20) known IGFIIR mutation were examined, along with matching normal tissues. Results were statistically significant in the following categories: (a) decreased active TGF-beta1 protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues; (b) increased LTGF-beta1 protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues; and (c) increased IGFII ligand protein expression in IGFIIR-mutant tumor tissues versus matching normal tissues or IGFIIR-wild-type tumor tissues. These data suggest that in genetically unstable GI tumors, mutation of a microsatellite within the coding region of IGFIIR functionally inactivates this gene, causing both diminished growth suppression (via decreased activation of TGF-beta1) and augmented growth stimulation (via decreased degradation of the IGFII ligand).

Transforming growth factor-betas block cytokine induction of catalase and xanthine oxidase mRNA levels in cultured rat cardiac cells.

We examined the effects of transforming growth factor-beta (TGF-beta) on the mRNA expression of the antioxidative enzymes, catalase, manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD), as well as the oxidative enzyme, xanthine oxidase (XO), in cultures of cardiomyocytes, cardiac non-myocytes, and fetal bovine heart endothelial cells. TGF-betas alone had little effect on expression of these enzymes, but treatment with a combination of interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha increased expression of MnSOD, catalase, and XO in some cell types with little effect on CuZnSOD expression. When TGF-betas were added along with these inflammatory cytokines there was a return to control levels of catalase expression, as well as a dramatic reduction in XO expression. In fetal bovine heart endothelial cells, treatment with inflammatory cytokines increased XO mRNA expression 11.5-fold and inclusion of TGF-betas reduced this 4-5-fold: effects on XO enzyme activity paralleled those seen on mRNA expression. Similar changes in XO expression were seen in cardiomyocytes. In contrast, TGF-betas did not change cytokine-induced MnSOD expression. All three mammalian isoforms of TGF-beta showed similar effects. In summary, TGF-betas may be able to decrease superoxide anion production and subsequent tissue damage by decreasing levels of XO.