Autocrine role of transforming growth factor beta1 on rat granulosa cell proliferation

We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.     

Altered secretion and processing of epidermal growth factor in adrenergic-induced growth of the rat submandibular gland

The granular convoluted tubule (GCT) cells of the submandibular glands represent a major production site for epidermal growth factor (EGF). This study investigates EGF production in the submandibular glands in relation to beta-adrenergic stimulation. Rats were treated with isoproterenol (beta-agonist), which caused up to a 400% increase in submandibular tissue weight after 3 weeks. The weight increase coincided with marked morphologic changes, with degranulation and an apparent decrement in the number of the GCT cells. Immunostaining against EGF revealed a reduction in the number of EGF-immunoreactive cells. Concomitantly, the glandular contents of 6-kDa EGF decreased from 12.86+/-3.42 nmol/gland (mean+/-S.E.M.) in controls to 0.26+/-0.03 nmol/gland. EGF mRNA levels, expressed relative to total RNA levels, only tended to be reduced after 3 weeks as judged from RT-PCR and in situ hybridization (ISH). The isoproterenol-treated rats had increased output of EGF in the saliva, but the salivary secretion of protein was also increased. In both glandular tissue and saliva, gel filtration revealed partially processed high molecular weight forms of EGF in the isoproterenol-treated rats. These data indicate that isoproterenol treatment leads to a hyperstimulatory state of the GCT cells, which then causes depletion of the cellular stores of mature EGF, and most likely due to a shortened posttranslational transit, incomplete peptide processing.     

Expression of transforming growth factor beta 2 RNA during murine embryogenesis

We have studied the temporal and spatial expression of transforming growth factor beta 2 (TGF beta 2) RNA in mouse embryos from 10.5 days post coitum (p.c.) to 3 days post partum (p.p.) by in situ hybridization analysis. TGF beta 2 RNA is expressed in a variety of tissues including bone, cartilage, tendon, gut, blood vessels, skin and fetal placenta, and is in general found in the mesenchymal component of these tissues. The expression of TGF beta 2 RNA changes during development in a manner consistent with a role for the gene product in mediating mesenchymal-epithelial interactions.     

Renal transforming growth factor beta 1 production in neonate rats

Abstract. The newborn rat kidney is not fully developed until approximately 12 days after birth. In order to evaluate the possible role of Transforming Growth Factor beta 1 (TGF beta 1) in renal development we analyzed the mRNA level for TGF beta 1 in sixty Wistar rats aged 1,15 and 30 days. We also performed immunohislochemical studies to visualize the distribution of this peptide in the kidney of these rats using a TGF beta 1 antibody. The results show that the mRNA levels for TGF beta 1 are higher in the kidneys of the 1-day-old rats than in the 15 (1.4 fold) and 30-day-old rats (1.7 fold). The immunohistochemical reaction revealed the presence of TGF beta in the kidneys of the rats. The staining intensity was higher in the renal cortex than in the medulla. The data suggests that TGF beta may be important during kidney development.     

Expression of epidermal growth factor in the kidney and submandibular gland during mouse postnatal development

Earlier work has demonstrated that the salivary glands and kidneys are the major sites of epidermal growth factor (EGF) synthesis in adult mice. The precise timing of the onset of endogenous EGF synthesis in these tissues is not yet clear. In the present study we assessed the ontogenesis of EGF expression in the Swiss-Webster mouse. Paraformaldehyde-fixed frozen sections of neonatal kidneys and salivary glands were probed with proEGF cRNA labelled with 35S for in situ hybridization and with rabbit antisera to mouse EGF for immunocytochemistry. Both EGF mRNA and immunoreactivity were first detected in the developing distal nephron between days 3 and 5 postpartum. Juxtamedullary nephrons underlying the superficial nephrogenic zone were the first to express EGF. During the 2nd week after birth, EGF-expressing tubules became more abundant and distributed to medullary as well as cortical regions, corresponding to the thick ascending limb of Henle and distal convoluted tubule. Initial EGF mRNA and immunoreactivity in the submandibular gland were first detected between days 18 and 20 postpartum and increased notably during the following weeks.     

Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects

The cytokine-transforming growth factor beta1 (TGFB1) is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We compared mammary gland morphogenesis in virgin Tgfb1(+/+), Tgfb1(+/-), and Tgfb1(-/-) mice and transplanted Tgfb1(+/+) and Tgfb1(-/-) epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated relative to the timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly, irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1(-/-) mice. The terminal end buds appeared to be normal morphologically, and a comparable amount of epithelial proliferation was evident. When transplanted into wild-type recipients, however, Tgfb1(-/-) epithelium showed accelerated invasion compared with Tgfb1(+/+) epithelium. This suggests that the normal rate of ductal extension in Tgfb1(-/-) null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1(-/-) mice were morphologically similar to those in Tgfb1(+/+) and Tgfb1(+/-) animals, with a normal branching pattern, and the tissue differentiated into early alveolar structures in the diestrous phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis: autocrine inhibition of epithelial ductal growth, and endocrine or paracrine stimulation of epithelial ductal growth.     

Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.     

Modulation of transforming growth factor beta signalling pathway genes by transforming growth factor beta in human osteoarthritic chondrocytes: involvement of Sp1 in both early and late response cells to transforming growth factor beta

Introduction  

Transforming growth factor beta (TGFβ) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is particularly important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. While the action of TGFβ on chondrocyte metabolism has been extensively catalogued, the modulation of specific genes that function as mediators of TGFβ signalling is poorly defined. In the current study, elements of the Smad component of the TGFβ intracellular signalling system and TGFβ receptors were characterised in human chondrocytes upon TGFβ1 treatment.     

Expression of the alpha subunit of 7S nerve growth factor in the mouse submandibular gland

-NGF is an inactive serine protease that is associated in the mouse submandibular gland with a closely related serine protease, -NGF, and the neurotrophic factor, -NGF. The heterogeneity of purified -NGF has been examined by DEAE-cellulose chromatography and SDS polyacrylamide gel electrophoresis. A possible explanation for the observed heterogeneity is presented. Antibodies have been prepared against -NGF and purified by affinity chromatography so that they do not cross-react with -NGF. This antibody preparation recognizes two very similar proteins in male mouse submandibular gland RNA-directed cell-free translation mixtures. The expression of only one of these forms is regulated by testosterone. Oligonucleotide probes specific for each of the three NGF subunits have been prepared and used for Northern blot analysis of RNA from the mouse submandibular gland. The three subunits were found to be coordinately expressed and each were 30-fold more abundant in male than in female glands.Abbreviations used NGF nerve growth factor - -, -, and -NGF -, -, and -subunits of mouse 7S NGF - PBS phosphate buffered saline - DTT dithiothreitol - PPO 2,5-Diphenyloxazole - DMSO dimethylsulfoxide - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - SSC 0.15M NaCl, 15 mM sodium citrate Supported by USPHS research grant NS19964. This paper is respectfully dedicated to Profs. Eric M. Shooter and Silvio Varon in recognition of their many contributions to our understanding of the structure and function of nerve growth factor.     

Atrial natriuretic factor intracellular signaling in the rat submandibular gland

We previously reported that intravenously administered atrial natriuretic factor (ANF) induced no salivation but enhanced agonist-evoked secretion in submandibular glands. The gene expression of ANF and natriuretic peptide receptors (NPR) was later reported in the glands. In the present study we sought to establish the intracellular signalling mechanisms underlying ANF modulation of salivary secretion. Fasted rats were prepared with submandibular duct and femoral cannulation. Dose–response curves to methacholine (MC) and norepinephrine (NE) were performed in the presence of cANP (4–23 amide) (selective NPR-C agonist) and ANF. Local injection of the agonist or ANF-induced no salivation, but enhanced MC and NE-evoked secretion. ANF and cANP (4–23 amide) enhanced phosphoinositide turnover being the effect abolished by U73122 (PLC inhibitor). Further ANF and cANP (4–23 amide) decreased basal cAMP content but failed to affect isoproterenol or forskolin-evoked cAMP. ANF response was inhibited by pertussis toxin and mimicked by cANP (4–23 amide) strongly supporting NPR-C activation. ANF-induced cAMP reduction was abolished by PLC and PKC inhibitors. The content of cGMP was dose dependently stimulated by ANF but not modified by cANP (4–23 amide). These findings support that ANF through NPR-C receptors coupled to PLC activation and adenylyl cyclase inhibition interacts with sialogogic agonists in the submandibular gland to potentiate salivation.     

Role of transforming growth factor beta in cancer

Transforming growth factor beta (TGF-beta) is an effective and ubiquitous mediator of cell growth. The significance of this cytokine in cancer susceptibility, cancer development and progression has become apparent over the past few years. TGF-beta plays various roles in the process of malignant progression. It is a potent inhibitor of normal stromal, hematopoietic, and epithelial cell growth. However, at some point during cancer development the majority of transformed cells become either partly or completely resistant to TGF-beta growth inhibition. There is growing evidence that in the later stages of cancer development TGF-beta is actively secreted by tumor cells and not merely acts as a bystander but rather contributes to cell growth, invasion, and metastasis and decreases host-tumor immune responses. Subtle alteration of TGF-beta signaling may also contribute to the development of cancer. These various effects are tissue and tumor dependent. Identifying and understanding TGF-beta signaling pathway abnormalities in various malignancies is a promising avenue of study that may yield new modalities to both prevent and treat cancer. The nature, prevalence, and significance of TGF-beta signaling pathway alterations in various forms of human cancer as well as potential preventive and therapeutic interventions are discussed in this review.     

Decrease in transforming growth factor beta 1 binding during differentiation of rat adipocyte precursors in primary culture

Transforming growth factor beta 1 (TGF-beta 1) binding and action were investigated during differentiation of adipocyte precursors freshly isolated from rat inguinal fat-pad cultivated in defined medium. The data presented in this paper indicate that TGF-beta 1 inhibits differentiation of adipocyte precursors with a 50% effective dose of 9 pM. Time course experiments demonstrate that TGF-beta 1 is active only when it is added to the cells while they are still undifferentiated. If added after the cells have started to differentiate, TGF-beta 1 is less active or becomes inactive. 125I-TGF-beta 1 binding studies on adipocyte precursors before and after differentiation indicate a 10-fold decrease in the number of TGF-beta 1 binding sites after the cells have differentiated. Blocking of the differentiation process by treating the cells with fetal bovine serum or with prostaglandin F2 alpha prevented the decrease in the number of TGF-beta 1 receptors, thereby demonstrating that this change in binding was specifically linked to the differentiation process. Experiments cross-linking 125I-TGF-beta 1 to adipocyte precursors showed that 125I-TGF-beta 1 is specifically cross-linked to two bands with molecular weights of 92,000 and 70,000. After differentiation, a decrease in the intensity of the cross-linked bands was observed. These results demonstrate that loss of cell surface TGF-beta 1 binding sites follows differentiation of adipocyte precursors.     

Ethanol disrupts cell cycle regulation in developing rat cortex interaction with transforming growth factor beta1

Ethanol is a potent teratogenic agent that disrupts several aspects of neuronogenesis, including the proliferation rate of cortical precursors. With regard to corticogenesis, possible targets of ethanol toxicity include soluble factors, like transforming growth factor beta1 (TGFbeta1), that regulate cortical growth and cell cycle proteins that control the kinetics of the cell cycle. The effect of ethanol on normal cell proliferation and TGFbeta1-regulated cell proliferation in the developing cortex was assessed using an organotypic slice culture model. Ethanol elongated the cell cycle, possibly through a decrease in the expression of G1 cell cycle protein cyclin D1. Further, ethanol exposure antagonized the anti-proliferative action of TGFbeta1 and blocked TGFbeta1-dependent increases in cell cycle inhibitor p21. Collectively, this evidence suggests that disruption of appropriate cell cycle protein expression and inhibition of TGFbeta1 activity are potential mechanisms underlying the effect of ethanol on cortical development.     

Expression of transforming growth factor beta (TGF-b1) by human preterm lung inflammatory cells

Using a previously published model of human BPD this study examines whether preterm lung inflammatory cells produce transforming growth factor beta 1 (TGF-beta1), a cytokine pivotal in pathogenesis of bronchopulmonary dysplasia (BPD), and whether TGF-beta1 expression is regulated by inflammation. Lung inflammatory cells (neutrophils and macrophages) recovered in the broncho-alveolar (BAL) fluid of premature infants intubated for respiratory distress after birth expressed TGF-b1 mRNA and protein. Total and bioactive TGF-beta1 were abundantly found in the BAL fluid of the same infants. In cell culture stimulation by lipopolysaccharide (LPS) did not result in any further expression of total or bioactive TGF-beta1 by neonatal lung inflammatory cells over constitutive concentrations. In conclusion, lung inflammatory cells from premature infants are a source of TGF-beta1 but LPS does not regulate TGF-b1 production in these cells.