Transforming growth factor-beta inhibits progesterone-induced enkephalinase expression in human endometrial stromal cells

The specific activity of enkephalinase in endometrial tissue of nonpregnant ovulatory women is correlated in a highly significant, positive manner with the plasma level of progesterone. The specific activity and levels of enkephalinase messenger ribonucleic acid and immunoreactive protein also are increased in human endometrial stromal cells in culture by treatment with a synthetic progestin, medroxyprogesterone acetate (MPA), in a time- and dose-dependent manner. From an analysis of the temporal relationship between the specific activity and half-life of enkephalinase in endometrial tissue and the level of progesterone in plasma, it appeared highly likely that some mechanism, in addition to progesterone withdrawal, was operative to reduce enkephalinase activity in endometrium during the late luteal phase of the ovarian cycle before progesterone levels had declined below those known to be effective for progesterone action. In stromal cells previously (and concurrently) treated with MPA (10(-9) mol/L), the addition of transforming growth factor-beta 1 (TGF beta 1) or TGF beta 2 (1 ng/mL) to the medium caused a decrease in enkephalinase specific activity despite the continued presence of MPA. The half-life of enkephalinase (activity) in stromal cells treated with MPA plus TGF beta 1 was 2.8 days, which is similar to the computed half-life for enkephalinase in endometrial tissue during the mid- to late secretory phase of the endometrial cycle (2.5 days). Simultaneous treatment of endometrial stromal cells with MPA (10(-9) mol/L) and TGF beta 1 (1 ng/ mL) prevented the progestin-induced increase in enkephalinase specific activity and immunoreactive enkephalinase protein. Thus, TGF beta acts to oppose the progesterone-induced increase in enkephalinase expression in endometrial stromal cells, even in the continued presence of MPA.     

Transforming growth factor-beta 3

Transforming Growth Factor-Beta (TGF-beta) is the general name for a family of naturally-occurring polypeptides which have multiple regulatory effects on cell proliferation and differentiation. Over the last decade it has become apparent that TGF-betas can be produced by most cell types and exert a wide range of effects in a context-dependent autocrine, paracrine or endocrine fashion via interactions with distinct receptors on the cell surface. This review summarizes current knowledge concerning the molecular and cellular biology of TGF-beta 3, the most recently described mammalian isoform, and focuses on those physiological actions which may lead to clinical applications, particularly in the indication areas of wound healing and chemoprotection.     

Reinforced cytotoxicity of lymphokine-activated killer cells toward glioma cells by transfection with the tumor necrosis factor-alpha gene

Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human tumor necrosis factor (TNF)-alpha gene by means of novel liposomes with a positive change on their surface. The cells secreted significant amounts of TNF-alpha into the culture medium and exhibited reinforcement of cytotoxicity toward a human glioma cell line (U251-SP), being three times more cytotoxic than nontransfected LAK cells. The mechanism for the reinforcement of cytotoxicity is considered to involve not only an increase in TNF-alpha secretion from LAK cells but also its expression on their surface. Intratumoral or intrathecal injection of LAK cells transfected with the TNF-alpha gene may be useful for the treatment of patients with malignant gliomas.     

Transforming growth factor-beta1 stimulates protein kinase A in mesangial cells

We recently demonstrated that transforming growth factor-beta (TGF-beta) stimulates phosphorylation of the type I inositol 1,4, 5-trisphosphate receptor (Sharma, K., Wang, L., Zhu, Y., Bokkala, S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine mesangial cells. In the present study, we evaluated whether TGF-beta stimulates PKA activation. Utilizing a specific PKA kinase assay, we found that TGF-beta increases PKA activity by 3-fold within 15 min of TGF-beta1 treatment, and the enhanced kinase activity was completely reversed by the inhibitory peptide for PKA (PKI; 1 microM). In mesangial cells transfected with a PKI expression vector, enhanced PKA activity could not be demonstrated with TGF-beta1 treatment. TGF-beta1 was also found to stimulate translocation of the alpha-catalytic subunit of PKA to the nucleus by Western analysis of nuclear protein as well as by confocal microscopy. TGF-beta1-mediated phosphorylation of cAMP response element-binding protein was completely reversed by H-89 (3 microM), a specific inhibitor of PKA. Stimulation of fibronectin mRNA by TGF-beta1 was also attenuated in cells overexpressing PKI. We thus conclude that TGF-beta stimulates the PKA signaling pathway in mesangial cells and that PKA activation contributes to TGF-beta stimulation of cAMP response element-binding protein phosphorylation and fibronectin expression.     

Ability of lymphokine-activated killer cells to lyse mycobacteria-infected cells

In cats and monkeys, we examined the parasympathetic component of the oculomotor complex, which directly innervates the ciliary muscle, using horseradish peroxidase (HRP). Labeled neurons of varying form and size were found in the Edinger-Westphal(EW) and the Perlia nuclei of the cat and in the anteromedian, EW, and Perlia nuclei of the monkey. Our study confirmed that a direct parasympathetic pathway exists from the midbrain to the ciliary muscles, and that accommodation is controlled in part by this direct link from the midsagittal region via a parasympathetic neuron of the oculomotor nuclear complex.     

Transforming growth factor-beta: a general review

Three isoforms of Transforming Growth Factor-beta (TGF-beta 1, beta 2 and beta 3) exist in mammals. They play critical roles in growth regulation and development. Each isoform is encoded by a unique gene on different chromosomes. All three of these growth factors are secreted by most cell types, generally in a latent form, requiring activation before they can exert biological activity. This activation of latent TGF-beta, which may involve plasmin, thrombospondin and possibly acidic microenvironments, appears to be a crucial regulatory step in controlling their effects. The TGF-betas possess three major activities: they inhibit proliferation of most cells, but can stimulate the growth of some mesenchymal cells; they exert immunosuppressive effects; and they enhance the formation of extracellular matrix. Two types of membrane receptors (type I and type II) possessing a serine/threonine kinase activity within their cytoplasmic domains are involved in signal transduction. Inhibition of growth by the TGF-betas stems from a blockage of the cell cycle in late G1 phase. Among the molecular participants concerned in G1-arrest are the Retinoblastoma (Rb) protein and members of the Cyclin/Cyclin-dependent kinase/Cyclin dependent kinase inhibitor families. In the intact organism the TGF-betas are involved in wound repair processes and in starting inflammatory reactions and then in their resolution. The latter effects of the TGF-betas derive in part from their chemotactic attraction of inflammatory cells and of fibroblasts. From gene knockout and from overexpression studies it has been shown that precise regulation of each isoform is essential for survival, at least in the long term. Several clinical applications for certain isoforms have already shown their efficacy and they have been implicated in numerous other pathological situations.     

Increased sensitivity of gastric cancer cells to natural killer and lymphokine-activated killer cells by antisense suppression of N-acetylgalactosaminyltransferase

UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) catalyze the initial reaction in O-linked (mucin type) oligosaccharide biosynthesis. To attempt to inhibit the synthesis of O-glycan, we transfected antisense cDNA of GalNAc transferase type 1 (GalNAc-T1) into a human gastric cancer cell line, JRST. A decreased expression of GalNAc-T1 at the level of both mRNA and protein was observed in the resultant transfectants. They demonstrated a significantly increased sensitivity to NK and lymphokine-activated killer cells in vitro compared with parental cells and mock transfectants. Although there was no significant difference in in vitro cell proliferation among parental cells, mock transfectants, or antisense transfectants, the in vivo growth rate of antisense transfectants using SCID mice was clearly lower than that of parental cells and mock transfectants. Furthermore, the treatment of mice with anti-asialo-G(M1) Ab abolished this growth-inhibitory effect of antisense transfection. From these results, we conclude that antisense suppression of GalNAc-T1 could increase the sensitivity of tumor cells to NK and lymphokine-activated killer cells, suggesting that this strategy may be of use as a new immunogene therapy.     

Transforming growth factor-beta expression in mouse lung carcinogenesis

Transforming growth factor-beta (TGF-beta) is a multifunctional growth modulator that inhibits the proliferation of many epithelial cells while stimulating the proliferation of most fibroblasts. To examine the role of TGF-beta in mouse lung chemically induced tumorigenesis, expression of the TGF-beta 1, -beta 2, and -beta 3 proteins was examined in A/J mice treated with the carcinogen urethane to induce lung adenomas using immunohistochemical staining analysis. Immunostaining for the TGF-beta ligands was detected in the epithelium of the bronchioles of untreated A/J mice with immunostaining being more intense for TGF-beta 1 than for TGF-beta 2 and TGF-beta 3; immunostaining for each TGF-beta ligand was also detected in the bronchiolar epithelium of urethane-treated A/J mice at levels similar to untreated mice. Immunostaining for the TGF-beta ligands was also detected in adenomas by 2 months; staining for TGF-beta 1, -beta 2, and -beta 3 in adenomas was detected at levels comparable with bronchioles. Following treatment with urethane for 8 months, immunostaining for TGF-beta s 1, 2, and 3 in bronchioles persisted at levels comparable to that in normal bronchioles and also persisted in adenomas, with staining for the TGF-beta ligands being very prominent on the edge of the tumor. Expression of TGF-beta 1 mRNA was examined in urethane-treated mouse lung tissue using Northern blot hybridization; here, expression of TGF-beta 1 mRNA increased 2-fold in 3-month urethane-treated lung tissue and an additional 2.5-fold by 8 months following urethane administration. Expression of TGF-beta 1 mRNA was also examined in nontumorigenic and tumorigenic mouse lung cells; in these cells, expression of TGF-beta 1 mRNA was higher in the tumorigenic cells than in the nontumorigenic cell line. These data show that there is an increase in expression of TGF-beta 1 during tumorigenesis and suggest that TGF-beta may play an important role in mouse lung carcinogenesis induced by urethane.     

Transforming growth factor-beta enhances adhesion of melanoma cells to the endothelium in vitro

Melanoma invasion requires migration through the vascular barrier. An early event in this process is the adhesion of metastatic cells to the endothelium. To elucidate the role of TGF-beta in the regulation of this process, human melanoma SK-MEL24 cells were labelled with [5'-(3)H]-thymidine and co-cultured with bovine pulmonary artery endothelial-cell monolayers. Radioactivity was assumed to be proportional to the number of SK-MEL24 cells bound to the endothelium. A low number of melanoma cells adhered to endothelial cells in a time-related manner. Pretreatment for 24 hr with 0.001 to 10 ng/ml TGF-beta1 or TGF-beta2 of both cell types enhanced melanoma-endothelium adhesion in a dose-dependent manner. Both melanoma and endothelial cells expressed RI- and RII-type TGF-beta receptors. The effect of TGF-beta was abolished by co-incubation with the proteoglycan decorin. Conditioned media from melanoma-endothelium co-cultures contained latent TGF-beta and failed to affect cell-cell adhesion. However, activation of TGF-beta by heating the medium or reducing the pH, increased melanoma-endothelium adhesion to an extent similar to that of the TGF-beta administered to the cultures. Zimography demonstrated that both cell types expressed urokinase-type plasminogen activator (uPA). Addition of plasminogen to the co-cultures, which was likely to be activated to plasmin by uPA, resulted in activation of TGF-beta and parallel stimulation of melanoma-endothelium adhesion. In conclusion, TGF-beta may enhance adhesion of melanoma cells to the endothelium, playing a relevant autocrine/paracrine role in the progression of invasive melanoma.     

RRR-alpha-tocopheryl succinate inhibits DNA synthesis and enhances the production and secretion of biologically active transforming growth factor-beta by avian retrovirus-transformed lymphoid cells

The RRR-alpha-tocopheryl succinate form of vitamin E, referred to as vitamin E succinate (VES), inhibits the proliferation of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells in a dose-dependent manner in vitro. Analyses of conditioned medium (CM) from VES growth-inhibited cells revealed a potent antiproliferative activity. Characterization of the antiproliferative activity as transforming growth factor-beta (TGF-beta) was established by 1) growth inhibition of TGF-beta-responsive Mv1Lu mink lung and murine CTLL-2 cell lines, 2) a combination of physical characteristics including heat stability, acid stability, and Bio-Gel P-60 column chromatography elution profile, 3) neutralization of the antiproliferative activity by antibodies specific for TGF-beta, and 4) immunoprecipitation of metabolically labeled TGF-beta in CM from VES-treated C4-1 cells by use of TGF-beta-specific antibodies. Northern blot analyses of total cellular RNA revealed that VES does not alter the levels of constitutively expressed TGF-beta isoform-specific mRNAs; namely, VES does not alter the levels of the 3.9- and 4.1-kb TGF-beta 2 mRNAs, the 3.0-kb TGF-beta 3 mRNA, or the 2.5-, 2.7-, and 1.7-kb TGF-beta 4 mRNAs. The data show that VES inhibits C4-1 cell proliferation and induces the cells to produce and secrete active forms of TGF-beta, suggesting that one mechanism whereby VES inhibits C4-1 cell proliferation may be via the TGF-beta pathway for cellular growth control.     

Transforming growth factor-beta1 increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells

Osteoclastogenesis inhibitory factor (OCIF), also termed osteoprotegerin (OPG), is a secreted member of the tumor necrosis factor (TNF) receptor family. It inhibits bone resorption in vivo and osteoclast-like cell (OCL) formation in vitro. To better understand the biological role of OCIF, we first examined the effects of various osteotropic agents on OCIF mRNA levels in mouse calvarial osteoblasts. Northern blot analysis showed that stimulators of OCL formation such as 1,25-(OH)2D3, prostaglandin E2 (PGE2), parathyroid hormone (PTH), and interleukin 1 (IL-1) decreased OCIF mRNA levels. In contrast, transforming growth factor (TGF)-beta1 increased OCIF mRNA levels in primary osteoblasts as well as in osteoblastic/stromal cell lines. Since it was reported that both TGF-beta1 and OCIF not only inhibited OCL formation but also impaired the survival of OCL by inducing apoptosis in vitro, we next examined the possible involvement of OCIF in TGF-beta1-induced impairment of OCL survival. In a mouse bone marrow culture, we confirmed that addition of OCIF or TGF-beta1 decreased the number of surviving OCL. Anti-OCIF IgG, which completely neutralized the effect of OCIF, partially prevented the TGF-beta1-induced decrease in the number of OCL. Our results suggest that (i) downregulation of OCIF expression is one of the mechanisms for the stimulatory effects of 1,25(OH)2D3, PGE2, PTH, and IL-1 on osteoclastogenesis; and (ii) the TGF-beta1-induced apoptosis of OCL is mediated, at least in part, by upregulation of OCIF expression.     

Transforming growth factor-beta1 in hemangioma of the ovary

OBJECTIVE: Anastomotic pseudoaneurysms continue to be a late complication of vascular surgery, particulary following prosthetic graft procedures. The purpose of this study was to investigate if a previously reported increase in interval between the original operation and the development of pseudoaneurysm was related to a change in incidence. DESIGN: Retrospective study. METHODS: We reviewed the records of 76 patients who presented with 90 femoral pseudo-aneurysms and underwent reconstructive procedures from January 1989 to June 1994. The median age was 69 years (range: 39-83). In the same time period all femoral artery anastomosis operations were recorded. RESULTS: The incidence of femoral pseudo-aneurysms in Copenhagen was approximately 4.3%. CONCLUSIONS: A previously reported increase in interval between primary operation and aneurysms formation was not related to a change in incidence during the same time period.     

Porcine interferon-gamma inhibits the growth of Legionella pneumophila in WiREF cells in vitro

In the present study eight human eyeballs were specifically prepared for scanning-electron-microscopic observation of the zonule. The zonule consisted of two main layers of radial fibres, an anterior and a posterior one, that inserted on the anterior and the posterior lens capsules, respectively. Some fibres inserted on the equator of the lens. Posterior zonular fibres originated at the pars plana, entered the dorsal part of the ciliary valleys and then changed their direction towards the posterior face of the lens. Posterior fibres inserted on the posterior capsule of the lens by branched endings 1 mm behind the equator of the lens. Anterior zonular fibres originated mainly at the pars plana and occasionally at the ciliary valleys. After running completely through the ciliary valleys in close contact with the lateral walls of the ciliary processes, they changed their direction at the anterior endings of the pars plicata and reached the anterior lens capsule. Anterior zonular insertions were achieved by webbed endings that diffused into the anterior capsule 2 mm in front of the lens equator. The extraordinary distension capacity of the zonular fibres was demonstrated by pulling the anterior lens capsule after hydrodissection. As a consequence, the anterior fibres were stretched up to four times their original length without breaking or disinserting.     

Probenecid inhibits transforming growth factor-beta 1 induced pyrophosphate elaboration by chondrocytes

OBJECTIVE: The elaboration of excess extracellular inorganic pyrophosphate (ePPi) by cartilage contributes to calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. Transforming growth factor-beta 1 (TGF beta 1) is the only defined physiologic stimulant of cartilage ePPi elaboration. The mechanism of ePPi generation by chondrocytes is unknown, but current evidence suggests that TGF beta 1 induced ePPi is made intracellularly. An active transport mechanism such as an anion transporter would then be necessary to export ePPi to the matrix where crystals form. We determined the effect of probenecid (PB), an anion transport inhibitor, on TGF beta 1 induced ePPi elaboration. METHODS: Porcine hyaline articular chondrocytes in high density monolayer cultures were exposed to serum-free media with and without TGF beta 1 and/or PB. ePPi was measured in the media after 48-96 h of exposure. Cell injury was measured by examining the release of 3H-deoxyglucose from chondrocytes. The activity of the ePPi generating ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH) and media lactate concentrations were measured with standard colorimetric assays. As PB may inhibit phosphodiesterase (PDE), its effects on ePPi generation were compared with isobutylmethylxanthine (IBMX), a specific PDE inhibitor. RESULTS: PB inhibited TGF beta 1 induced ePPi elaboration by chondrocytes. PB did not cause membrane injury or decrease NTPPPH activity. Lactate production was decreased by PB but did not correlate with the effects of PB on ePPi elaboration. IBMX did not inhibit TGF beta 1 effect on ePPi elaboration. CONCLUSION: PB blocks TGF beta 1 induced ePPi elaboration. This effect is independent of cell membrane injury, decreased NTPPPH activity, or PDE inhibition. Our data implicate a role for anion transport in TGF beta 1 induced ePPi elaboration, and suggest a potential therapy for CPPD disease.     

Transforming growth factor-beta isoforms in the developing chicken intestine and spleen: increase in transforming growth factor-beta 4 with coccidia infection

Expression of transforming growth factor-betas 2, 3 and 4 (TGF-beta) in the developing chicken intestine and spleen was investigated using specific cDNA probes and antibodies for the different TGF-beta isoforms. Coordinate expression of the mRNAs for TGF-beta s 2, 3 and 4 was detected in the embryonic intestine by 8 days, with maximal expression of the mRNAs for TGF-beta s 2 and 4 occurring at 12 and 19 days, respectively, while expression of TGF-beta 3 mRNA remained constant during this time. While specific antibodies for TGF-beta s 2, 3 and 4 could detect only weak immunohistochemical staining of the intestinal epithelium in 4-, 12- and 16-day-old embryos, intense staining for TGF-beta s 2, 3 and 4 was detected in the tips of the intestinal villi of 19-day-old embryos. In the spleen, expression of the mRNAs for TGF-beta s 2 and 3 increased in the newly hatched chick compared with the embryo and then decreased in the adult to levels that were lower than in the embryo; expression of TGF-beta 4 mRNA increased progressively with developmental age, with expression in the adult spleen being significantly higher than in the embryonic and hatchling spleen. Immunohistochemical staining of spleens showed a selective increase in the level of reactive TGF-beta 4 with increasing developmental age, while staining for TGF-beta s 2 and 3 was constant during development. After infection of 1-month-old chickens with coccidian parasite, expression of TGF-beta 4 mRNAs increased 5-8-fold in intestinal intra-epithelial lymphocytes and 2.5-fold in spleen cells, while expression of the mRNAs for TGF-beta s 2 and 3 remained constant in these cells. The results of this study suggest that TGF-beta may play a role in development of the intestine and spleen in the chicken and that TGF-beta 4 in particular increases after infection of coccidia in the chicken.     

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta1 (TGF-beta1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-beta1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-beta1 on the melanogenic activity of B16/F10 cells.