Bidirectional effects of Kupffer cells on hepatocyte proliferation in vitro.

The control of rat hepatocyte DNA synthesis in vitro by Kupffer cells and transformed perisinusoidal lipocytes, i.e. myofibroblast-like cells was studied. Conditioned media from Kupffer cells inhibit the replicative (hydroxyurea-sensitive) DNA synthesis dose-dependently in primary cultures of hepatocytes stimulated by epidermal growth factor (EGF). The cytokine responsible for the inhibition was identified as transforming growth factor beta (TGF beta). After neutralization of activated TGF beta in these media, DNA synthesis is stimulated in quiescent hepatocytes via transforming growth factor alpha (TGF alpha) demonstrated by competitive TGF alpha/EGF-receptor blocking on hepatocytes. Results similar to those obtained with Kupffer cells were found with conditioned media of myofibroblast-like cells. Northern blot hybridization confirms the expression of both TGF beta and TGF alpha in Kupffer cells and myofibroblast-like cells, respectively. These findings support the notion that Kupffer cells and myofibroblast-like cells might regulate in both directions liver regeneration depending on the proportion of secreted TGF alpha and TGF beta and on the activation status of TGF beta, of which a significant fraction is secreted in the latent form.     

Transforming growth factors (TGF alpha and TGF beta 1) stimulate chondroitin sulfate and hyaluronate synthesis in cultured rat liver fat storing cells

The synthesis of total sulfated glycosaminoglycans (GAG) was stimulated by transforming growth factors (TGF alpha 1.4-fold at 5 ng/ml, and TGF beta 1 2.05-fold at 2.5 ng/ml) in primary cultures of rat liver fat storing cells (FSC). The combination of both TGFs resulted in an additively stimulated synthesis of total sulfated GAG (more than 3-fold), chondroitin sulfate (more than 15-fold) and hyaluronate (3.8-fold), respectively, whereas the formation of dermatan sulfate was unchanged and that of heparan sulfate was slightly reduced. In summary, TGFs were identified as important mediators of stimulated GAG synthesis in those cells of the liver (FSC), which are the primary site of matrix glycoconjugate production.     

Growth regulation of ovarian cancer cells by epidermal growth factor and transforming growth factors alpha and beta 1.

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGF alpha and TGF beta 1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGF alpha stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF beta 1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF beta 1 and EGF/TGF alpha was antagonistic in OVCAR-3 cells. In contrast, EGF/TGF alpha and TGF beta 1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGF alpha, TGF beta 1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.     

Anti-sense transforming growth factor alpha oligonucleotides inhibit autocrine stimulated proliferation of a colon carcinoma cell line.

Many carcinoma cells secrete transforming growth factor alpha (TGF alpha). A 23 base anti-sense oligonucleotide that recognizes the TGF alpha mRNA inhibits both DNA synthesis and the proliferation of the colon carcinoma cell line LIM 1215. The effects of the anti-sense TGF alpha oligonucleotide are reversed by epidermal growth factor (EGF) at 20 ng/ml. When the LIM 1215 cells are grown under serum free conditions, the anti-sense TGF alpha oligonucleotides have their greatest effects at high cell density (2 x 10(5) cells/cm2), indicating that the secreted TGF alpha is acting as an exogenous growth stimulus. In addition, at higher cell densities, the kinase activity of the EGF receptor is activated and the receptor is down-modulated. The cell density dependent activation of the EGF receptor is inhibited by the application of the antisense TGF alpha oligonucleotides.     

Altered responses of regenerating hepatocytes to norepinephrine and transforming growth factor type beta

Norepinephrine (NE), acting through the alpha 1-adrenergic receptor, modules the response of rat hepatocytes in primary culture to transforming growth factor type beta 1 (TGF beta) by increasing the amount of TGF beta required for a given degree of inhibition of epidermal growth factor (EGF)-induced DNA synthesis (Houck et al., J. Cell. Physiol. 135:551-555, 1988). This effect was also found in hepatocytes isolated from regenerating livers but was greatly magnified in cells isolated between 12 and 18 hr after two-thirds partial hepatectomy (PHX). During this period of enhanced sensitivity, NE was equally potent in terms of dose but more efficacious in the regenerating hepatocytes. As it did in control hepatocytes (Cruise et al., Science 227:749-751, 1985), the alpha 1-adrenergic receptor mediated the activity of NE in regenerating hepatocytes. Vasopressin (VP) and angiotensin-II (AG) also antagonized the effect of TGF beta and showed increased activity in regenerating hepatocytes but at only 50% or less of the maximal effect reached by NE. Regenerating hepatocytes isolated 24-72 hr after PHX exhibited decreased sensitivity to inhibition by TGF beta, with a nadir in 48-hr-regenerating cells. These findings suggest that NE may be involved in triggering the early phase of DNA synthesis during liver regeneration, with the subsequent acquisition of innate resistance to TGF beta responsible for continued proliferation at a time when TGF beta mRNA is known to be increasing in the liver (Braun et al., Proc. Natl. Acad. Sci. USA 85:1539-1543, 1988). EGF induced increased DNA and protein synthesis in cultures of control hepatocytes; TGF beta inhibited the EGF-induced DNA synthesis but had no effect on protein synthesis. This may be relevant to the latter stages of liver regeneration, when high levels of TGF beta mRNA are detected in liver and cellular hypertrophy predominates over hyperplasia.     

Loss of TGF-beta dependent growth control during HSC transdifferentiation

Liver injury induces activation of hepatic stellate cells (HSCs) comprising expression of receptors, proliferation, and extracellular matrix synthesis triggered by a network of cytokines provided by damaged hepatocytes, activated Kupffer cells and HSCs. While 6 days after bile duct ligation in rats TGF-beta inhibited DNA synthesis in HSCs, it was enhanced after 14 days, indicating a switch from suppression to DNA synthesis stimulation during fibrogenesis. To delineate mechanisms modulating TGF-beta function, we analyzed crosstalk with signaling pathways initiated by cytokines in damaged liver. Lipopolysaccharide and tumor necrosis factor-alpha enhanced proliferation inhibition of TGF-beta, whereas interleukin-6, oncostatin M, interleukin-1alpha, and interleukin-1beta did not. Hepatocyte growth factor (HGF) counteracted TGF-beta dependent inhibition of DNA synthesis in quiescent HSCs. Since expression of c-met is induced during activation of HSCs and HGF is overrepresented in damaged liver, crosstalk of HGF and TGF-beta contributes to loss of TGF-beta dependent inhibition of DNA synthesis in HSCs.     

Transforming growth factor beta 1 and adrenocorticotropin differentially regulate the synthesis of adrenocortical cell heparan sulfate proteoglycans and their binding of basic fibroblast growth factor.

Adrenocortical differentiated functions are under the control of both endocrine hormones such as ACTH and local factors such as transforming growth factor beta (TGF beta) or basic fibroblast growth factor (bFGF). Besides their regulatory actions on the synthesis of corticosteroids, these two classes of factors also exert some important effects on the cellular environment. We have examined here the regulation by ACTH and TGF beta of adrenocortical cell proteoglycan synthesis and secretion. Under basal conditions, adrenocortical cells synthesized and secreted several species of sulfated proteoglycans, 80% of them being recovered in solution in the culture medium. When analyzed by ion exchange chromatography, the cell extracts and the media from cells metabolically labeled with 35S-sulfate were found to contain two and three species of radioactive sulfated proteoglycans, respectively. All species were proteoheparan-sulfates. Treatment of adrenocortical cells with TGF beta 1 or ACTH resulted in a significant increase of the incorporation of 35S into both secreted and cell-associated proteoglycans. ACTH stimulated more than three times the amount of secreted proteoglycans eluting from DEAE-Trisacryl as peak B, whereas TGF beta preferentially increased the amount of peak C. No important modification of the size of the synthesized proteoglycans was observed. The subpopulation of heparan sulfate proteoglycans capable to bind bFGF was also largely increased after ACTH or TGF beta treatment and paralleled the variation in overall proteoheparan sulfate synthesis. Thus those effects of TGF beta and ACTH on proteoglycan synthesis may participate in an increased ability of adrenocortical cells to bind and respond to bFGF.     

The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.     

Active synthesis of glycosaminoglycans by liver parenchymal cells in primary culture

Rat liver parenchymal cells were evaluated after 2 days of primary culture for their ability to synthesize and accumulate heparan sulfate as the major component and low-sulfated chondroitin sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. The newly synthesized glycosaminoglycans secreted into the medium were different from those remaining with and/or on the cell layer. Low-sulfated chondroitin 4-sulfate, a major glycosaminoglycan in blood, was synthesized in the order of 320 μg/liver per day, more than 90% of which was secreted into the medium within 16 h and 40% of the glycan secreted was degraded during that time. On the other hand, heparan sulfate, the major glycosaminoglycan synthesized by the parenchymal cells, was mainly distributed in the cell layer. After 8 days of culture, the synthesis of glycosaminoglycans by the cells increased markedly, especially dermatan sulfate, chondroitin sulfate and hyaluronic acid.     

TGF‐β Stimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways

The enzyme chondroitin polymerizing factor (ChPF) is primarily involved in extension of the chondroitin sulfate backbone required for the synthesis of sulfated glycosaminoglycan (sGAG). Transforming growth factor beta (TGF‐β) upregulates sGAG synthesis in nucleus pulposus cells; however, the mechanisms mediating this induction are incompletely understood. Our study demonstrated that ChPF expression was negatively correlated with the grade of degenerative intervertebral disc disease. Treatment of nucleus pulposus cells with TGF‐β induced ChPF expression and enhanced Smad2/3, RhoA/ROCK activation, and the JNK, p38, and ERK1/2 MAPK signaling pathways. Selective inhibitors of Smad2/3, RhoA or ROCK1/2, and knockdown of Smad3 and ROCK1 attenuated ChPF expression and sGAG synthesis induced by TGF‐β. In addition, we showed that RhoA/ROCK1 signaling upregulated ChPF via activation of the JNK pathway but not the p38 and ERK1/2 signaling pathways. Moreover, inhibitors of JNK, p38 and ERK1/2 activity also blocked ChPF expression and sGAG synthesis induced by TGF‐β in a Smad3‐independent manner. Collectively, our data suggest that TGF‐β stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1 and the three MAPK signaling pathways. J. Cell. Biochem. 119: 566–579, 2018. © 2017 Wiley Periodicals, Inc.     

Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells

Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possibility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of alpha(v)beta(3) integrin. alpha(v)beta(3) integrin was expressed by activated, myofibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence alpha(v) subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These alpha(v)beta(3) antagonists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by activated stellate cells was reduced by the alpha(v)beta(3) antagonists, while matrix metalloproteinase-9 synthesis was enhanced. Stellate cells incubated with active recombinant matrix metalloproteinase-9 showed enhanced apoptosis, while cells treated with a synthetic inhibitor of this protease showed increased survival. Our studies suggest that alpha(v)beta(3) integrin regulates the fate of hepatic stellate cells. Degradation of alpha(v)beta(3) ligands surrounding activated stellate cells during resolution of liver fibrosis might decrease alpha(v)beta(3) integrin ligation, suppressing stellate cell proliferation and inducing a fibrolytic, matrix metalloproteinase-secreting phenotype that may prime stellate cells for apoptosis.     

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.     

Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors

When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.     

Effects of transforming growth factor beta, tumor necrosis factor alpha, interferon gamma and LIF-HILDA on the proliferation of acute myeloid leukemia cells

A group of polypeptide factors that regulate cell growth and differentiation has been tested for their biological activities on the growth and differentiation of leukemic cells isolated from patients with Acute Myeloid Leukemias (AML). The effects of Transforming Growth Factor beta 1 (TGF beta), Tumor Necrosis Factor alpha (TNF alpha), Interferon gamma (IFN gamma) and LIF-HILDA were compared on leukemic cells cultured in vitro for seven days. Spontaneously growing leukemic cells were selected in order to study either inhibition or enhancement of proliferation induced by these factors. Only TGF beta 1 was found to induce a clear inhibition of leukemic proliferation in all cases tested. Recombinant TNF alpha and IFN gamma were found to induce either inhibition or enhancement of the proliferation on separate specimens. Under the conditions of culture, it was not possible to document any effect of LIF-HILDA. Cell differentiation and cell maturation were documented studying the modulation of cell surface antigens. TGF beta did not modify antigen expression on the cells surviving after 3 days in culture. Both TNF alpha and IFN gamma were found to enhance the expression of adhesion molecules and to a lesser extent, the expression of some lineage associated antigens. No effect of LIF-HILDA on antigen modulation was documented in the cases tested. These data confirm that TGF beta is by itself a potent inhibitor of the myeloid leukemia cells proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth regulatory peptide production by human breast carcinoma cells

The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.     

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Transforming growth factor-beta modulation of glycosaminoglycan production by mesenchymal cells of the developing murine secondary palate

Development of the mammalian secondary palate requires proper production of the extracellular matrix, particularly glycosaminoglycans (GAGs) and collagen. Endogenous factors that regulate the metabolism of these molecules are largely undefined. A candidate for a locally derived molecule would be transforming growth factor beta 1 (TGF beta 1) by virtue of its potency as a modulator of extracellular matrix metabolism by several cell lines. We have thus attempted to assign a regulatory role for TGF beta 1 in modulation of GAG production and degradation by mesenchymal cells of the murine embryonic palate (MEPM). Treatment with TGF beta 1 or TGF beta 2, but not IGF-II, resulted in a stimulation of total GAG synthesis. Furthermore, cells treated with both TGF beta 1 and TGF alpha showed a synergistic increase in GAG synthesis if pretreated with TGF beta 1 but not TGF alpha. Simultaneous stimulation with TGF beta 1 and TGF beta 2 did not elicit a synergistic response. These studies demonstrate the ability of TGF beta, synthesized by embryonic palatal cells, to specifically stimulate GAG synthesis by MEPM cells. Other growth factors present in the developing craniofacial region may also modulate TGF beta-induced GAG synthesis, a biosynthetic process critical to normal development of the embryonic palate.