Dual functions for connexins: Cx43 regulates growth independently of gap junction formation.

Connexins, the family of proteins that form vertebrate gap junctions, have key roles during development and in the adult. Previously, the physiological actions of connexins have been ascribed solely to formation of gap junction channels and thought to be mediated by the transfer of small molecules between neighboring cells. In conflict with this hypothesis here we demonstrate that Cx43 can affect cell growth independently of gap junction formation. This conclusion is based on four findings: (1) There is a lack of correlation between the action of Cx43 mutants Cx43-S255A, Cx43-S279A, and Cx43-S282A on growth and cell coupling in 3T3 A31 fibroblasts. (2) Blockade of gap junction formation, by either heptan-1-ol treatment or culturing cells at low density, had no effect on the ability of the Cx43 mutants to control growth. (3) Wildtype Cx43 inhibited growth of Neuro2a cells under conditions where gap junctions were unable to form. (4) The CT domain of Cx43, which lacks intrinsic gap junction activity, is as effective as the wildtype molecule in suppressing the growth of Neuro2a cells. These observations demonstrate that Cx43 has dual functions: first, its well-accepted role in forming a gap junction channel and, second, a direct action of the connexin protein on growth that is mediated via the cytoplasmic carboxyl domain.

A connexin 43 mutant lacking the carboxyl cytoplasmic domain inhibits both growth and motility of mouse 3T3 fibroblasts.

Connexins have been shown to inhibit the growth of a wide number of communication-deficient cells both in vivo and in vitro, but the molecular mechanism remains largely unknown. In previous work we have shown that stable transfectants of 3T3 A31 fibroblasts, which express a Connexin 43 (Cx43) mutant (Cx43-256M) consisting of amino acids 1-256 of rat Cx43 fused to a c-myc tag, exhibit a decreased basal growth rate and weakened mitogenic response to platelet derived growth factor compared with either the parent cell line or cells transfected with an expression vector that did not encode a functional protein. Here we have investigated further the growth characteristics of these cells in order to establish the mechanism by which this protein suppresses cell growth. Analysis of DNA synthesis in individual cells by immunofluorescence staining of bromodeoxyuridine incorporation demonstrated that the slow growth of Cx43-256M cells was due to a decrease in the number of cells that undergo DNA synthesis following growth factor stimulation. This was associated with an increased proportion of the cell population in the G2/M phases of the cell cycle suggesting growth may be arrested during G2 or metaphase. In addition to effects on cell growth, Cx43-256M expression inhibited cell motility as assayed both in a wounding assay and in a Boyden chamber assay. These results now raise the question as to whether the primary effect of the Cx43-256M protein is on cell growth or cell motility.

Expression of a Cx43 deletion mutant in 3T3 A31 fibroblasts prevents PDGF-induced inhibition of cell communication and suppresses cell growth.

Communication through gap junctions was first suggested to have a role in the social control of cell growth over 30 years ago. However, despite extensive experimentation, the importance of gap junctions as a general mechanism of growth control remains to be established. A number of different studies have shown that a common early response of cells in culture to polypeptide growth factors such as PDGF is a rapid and transient inhibition of cell communication suggesting that a cell may have to lose communication with its neighbors before it can undergo cell division. Here we show that 3T3 A31 fibroblasts exposed to PDGF exhibit a 50% decrease in cell communication as measured by dye transfer in the absence of significant changes in the cellular content and distribution of Cx43. Likewise, PDGF inhibited cell communication in cells transfected either with a vector which did not contain a cDNA or with an expression vector encoding full-length Cx43 fused to a c-myc tag (Cx43-M). In contrast, 3T3 A31 fibroblasts transfected with an expression construct encoding a deletion mutant of Cx43 (Cx43-256M) consisting of amino acids 1-256 of Cx43 fused to a c-myc tag maintain high levels of gap junction activity following exposure to PDGF. These results suggest that sites which trigger loss of cell communication in response to PDGF are located within amino acids 257 to 382 of the Cx43 molecule. Cells transfected with an expression vector encoding full-length Cx43 fused to a c-myc tail exhibited a reduced basal growth rate compared to both parent cells and cells transfected with a control vector but maintained a strong mitogenic response to PDGF. In contrast, both the basal growth rate and the mitogenic response to PDGF was markedly reduced in cells which expressed Cx43-256M consistent with the hypothesis that loss of cell communication is required before a cell can respond to mitogenic stimuli.

DNA synthesis by ovine mammary alveolar epithelial cells: effects of heparin, epidermal growth factor-related peptides and interaction with stage of pregnancy.

Amphiregulin is a heparin-binding member of the epidermal growth factor (EGF) family, which we have recently shown to be expressed in sheep mammary gland. Uniquely among known EGF-like growth factors, its mitogenic activity is inhibited by soluble heparin, but heparin-like molecules on the cell surface and/or in extracellular matrix appear to be necessary for amphiregulin to exert its biological effect. In primary cultures of sheep mammary alveolar epithelial cells, heparin (1-20 mg/l) inhibited DNA synthesis in a dose-dependent manner. The extent of the inhibition was influenced by physiological state, being greater (P < 0.05) in mammary cell cultures derived from 5- to 10-week pregnant sheep (63.1 +/- 8.2%, mean +/- S.E.M., n = 8) than in cultures derived from sheep which were non-pregnant (35.8 +/- 8.3% inhibition, n = 6) or late, 20-week, pregnant (39.8 +/- 5.6%, n = 6). Both EGF and transforming growth factor-alpha (TGF-alpha) significantly (P < 0.001) increased DNA synthesis in the presence of heparin. The effect of TGF-alpha was dose-related, wholly reversing the inhibitory effect of heparin in cell cultures from non-pregnant and 20-week pregnant sheep. DNA synthesis was stimulated by amphiregulin and TGF-alpha increased the maximum response. The heparin antagonist, hexadimethrine, inhibited DNA synthesis, but, in the presence of amphiregulin, approximately equivalent concentrations of heparin overcame this inhibitory effect. In the presence of heparin, TGF-alpha showed synergistic interactions with insulin or IGF-I. The results indicate interactive effects of EGF and IGF growth factor families in sheep mammary growth.

HGF/SF inhibits junctional communication.

Hepatocyte growth factor/scatter factor (HGF/SF) is a fibroblast-derived protein that affects the growth, motility, and differentiation of epithelial and endothelial cells. We have investigated the effect of HGF/SF on junctional communication in mouse keratinocytes (MK cells). HGF/SF inhibited cell communication in MK cells as assessed by the transfer of a low-molecular-weight dye, Lucifer Yellow. The inhibition was rapid, the earliest effects being apparent 5 to 10 min after addition of the factor, and was transient. The decrease in dye transfer correlated with a loss of the gap junction protein connexin 43 as measured by Western blotting, probably due to increased protein degradation. The results show that junctional communication is an early target of HGF/SF activity and they are consistent with the hypothesis that gap junctions are primary targets of the action of growth factors.

Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.

Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.

Transforming growth factor-alpha: receptor binding and action on DNA synthesis in the sheep mammary gland.

Microsome factions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-alpha (TGF-alpha). Binding was dependent on microsomal protein concentration, time and temperature. It showed the characteristics of an epidermal growth factor (EGF) receptor, being displaced by TGF-alpha and EGF, but not by insulin or IGF-I. The non-linear curve fitting program LIGAND was used to determine affinity and number of binding sites. A single class of high-affinity binding sites was found. The apparent dissociation constant (Kd) was similar in all physiological states (2.43 +/- 0.27 mol/l x 10(-10), n = 23). Numbers of binding sites were lower in late-pregnant (20 weeks) and lactating sheep (14.07 +/- 2.45 fmol/mg protein, n = 10) than in non-pregnant, 10- or 15-week pregnant sheep (43.04 +/- 5.93 fmol/mg protein, n = 13). DNA synthesis by mammary alveolar epithelial cells cultured on collagen gels was increased twofold by TGF-alpha (maximum response at 10 micrograms/l; 1.8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-alpha on day 3 of culture, but the response was delayed to day 4-5 of culture in cells from other physiological states. Dose-response was not significantly affected. TGF-alpha and IGF-I produced an additive effect on DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta 1 and interleukin-1 beta stimulate LDL receptor activity in Hep G2 cells.

The effect of transforming growth factor-beta 1 (TGF-beta 1) and interleukin-1 beta (IL-1 beta) on LDL receptor in Hep G2 cells was investigated. A greater than two-fold stimulation of the binding and internalisation of [125I]-labelled LDL at 37 degrees C was observed after an 18-h incubation of the cells with TGF-beta 1 at 50 ng/ml and IL-1 beta at 11,700 units/ml compared with control cells. Scatchard analysis of the binding of [125I]-labelled LDL at 4 degrees C after an 18-h incubation of the cells with 1170 units/ml IL-1 beta and 5 ng/ml TGF-beta 1 showed that they were both acting primarily by increasing LDL receptor number. The increase in LDL receptor activity could not be attributed to an increase in cell proliferation as TGF-beta 1 at concentrations from 0.05 ng/ml to 50 ng/ml had no significant effect on either cell number or [3H]thymidine incorporation into DNA whilst IL-1 beta inhibited DNA synthesis by more than 80% at a concentration of 11,700 units/ml but had significant effect on cell number. Cholesterol biosynthesis from [14C]acetate, in contrast to the stimulation of LDL receptor activity, was inhibited by approximately two-fold by incubation with TGF-beta 1 at 50 ng/ml and IL-1 beta at 11,700 units/ml.

Porcine smooth muscle cell-conditioned medium stimulates LDL receptor activity in Hep G2 cells.

Paracrine factors may modulate low density lipoprotein (LDL) receptor activity in hepatocytes. To study this the effect of conditioned medium prepared from a range of cell types on the binding and internalisation of 125I-LDL in Hep G2 cells was studied. Seven of the fourteen conditioned media tested, including those from P388D1, U937, porcine smooth muscle (Pc SMC) Swiss 3T3, STO, = 48 and MDCK cells, were found to increase the binding and internalisation of 125I-LDL at 37 degrees C by Hep G2 cells (P < 0.01). The largest increase in LDL receptor activity was produced by conditioned medium from Pc SMC cells and was, therefore, selected for further analysis. The Pc SMC-conditioned medium increased LDL receptor number in Hep G2 cells by three-fold but had no effect on LDL receptor activity in human skin fibroblasts. DNA synthesis and cholesterol synthesis by Hep G2 cells were inhibited by Pc SMC-conditioned medium. Preliminary characterisation of the Pc SMC-derived factor(s) suggests that it is a protein(s) of low relative molecular mass.

Growth requirements and expression of LDL receptor and HMG-CoA reductase in Hep G2 hepatoblastoma cells cultured in a chemically defined medium.

A serum-free chemically defined medium (CDM) has been developed which sustains the growth in culture of the highly differentiated human hepatoma cell line Hep G2. Unlike rodent hepatoma lines, Hep G2 cells in serum-free medium have an absolute requirement for lipoprotein lipids (either low density lipoprotein (LDL) or high density lipoprotein (HDL)) for growth. In the presence of LDL (or HDL) growth was further enhanced by insulin, triiodo-L-thyronine, 17 alpha-ethinylestradiol but not by epidermal growth factor (EGF). On type I collagen gels cells cultured in CDM were contact inhibited and formed monolayers. This contrasted with the pattern of growth of cells cultured in the presence of serum on type I collagen gels and cells cultured on tissue-culture plastic in either CDM or medium containing serum which formed foci of multilayered cells. Expression of the LDL receptor and HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase genes was comparable in Hep G2 cells cultured in CDM and serum-containing medium. Furthermore, the binding and internalisation of 125I-LDL at 37 degrees C was modulated by hormones that have previously been shown to affect LDL receptor levels in liver in vivo or in hepatocytes cultured in serum-containing medium in vitro. The culture system described provides a basis for studying the regulation of hepatocyte-specific functions by soluble factors (either plasma- or cell-derived) and cell-substratum interactions in a human liver cell line.