黄芫花提取物对V79细胞和WB肝细胞的生物...：1....

A Chinese herb, wikstroemia Chamaedaphen (WC) extract, recently has been shown to be a potential tumor promoting agent on uterine cervical carcinoma induced by HSV-2 or MCA in mice. To determine whether the tumor promoting effects of WC extract were mediated through inhibition of gap junctional intercellular communication (GJIC) with relation to cellular growth, experiments were conducted on Chinese hamster V79 cells and rat WB liver cells by utilization of SLDT method for GJIC detection and cell growth curve examination, 3H-TdR incorporation, mitotic index (MI) and Flow Cytometry (FCM) methods. TPA was used for comparative purpose. WC extract inhibited GJIC and stimulated cell growth in a dose (2-200 micrograms/ml) and time (0-72 hr)-dependent manner in both cell lines. Both WC extract and TPA treatments increased V79 cell growth rate. The average cell doubling-time was decreased from 36.5 hr in control V79 cells to 28.2 hr in WC extract (10 micrograms/ml) and 20.9 hr in TPA (50 ng/ml) treatment by the 3rd day. Stimulating effect of both drugs on DNA synthesis of V79 cells was demonstrated. The results of FCM and MI indicated that the cell number of M-phase cells was increased after drug treatment. It is suggested that (1) tumor promoting effect of WC extract might be mediated through inhibition of GJIC: (2) inhibition of GJIC is closely correlated with increased cell growth rate and entry of cell division cycle.     

Characterization of a 60-kDa cell surface-associated transforming growth factor-β binding protein that can interfere with transforming growth factor-β receptor binding

We have characterized a 60-kDa transforming growth factor-β (TGF-β) binding protein that was originally identified on LNCaP adenocarcinoma prostate cells by affinity cross-linking of cell surface proteins by using ¹²⁵I-TGF-β1. Binding of ¹²⁵I-TGF-β1 to the 60-kDa protein was competed by an excess of unlabeled TGF-β1 but not by TGF-β2, TGF-β3, activin, or osteogenic protein-1 (OP-1), also termed bone morphogenetic protein-7 (BMP-7). In addition, no binding of ¹²⁵I-TGF-β2 and ¹²⁵I-TGF-β3 to the 60-kDa binding protein on LNCaP cells could be demonstrated by using affinity labeling techniques. The 60-kDa TGF-β binding protein showed no immunoreactivity with antibodies against the known type I and type II receptors for members of the TGF-β superfamily. Treatment of LNCaP cells with 0.25 M NaCl, 1 μg/ml heparin, or 10% glycerol caused a release of the 60-kDa protein from the cell surface. In addition, we found that the previously described TGF-β type IV receptor on GH3 cells, which does not form a heteromeric complex with TGF-β receptors, could be released from the cell surface by these same treatments. This suggests that the 60-kDa protein and the similarly sized TGF-β type IV receptor are related proteins. The eluted 60-kDa LNCaP protein was shown to interfere with the binding of TGF-β to the TGF-β receptors. Thus, the cell surface-associated 60-kDa TGF-β binding protein may play a role in regulating TGF-β binding to TGF-β receptors. J. Cell. Physiol. 173:447–459, 1997. © 1997 Wiley-Liss, Inc.     

Expression and function of gingival fibroblast C1q receptors are upregulated by interleukin-1β and transforming growth factor-β

In injury and inflammation, complement (C) component C1q, in addition to its central role in initiation of classical pathway of complement activation, modulates diverse cellular functions by binding to specific cell surface receptors. Interaction of substrate-bound C1q with receptors for the collagen-like domain of C1q (C1qRC) of human gingival fibroblasts (HGF) promotes cell attachment. We investigated modulation of the adhesive function and expression of C1qRC by interleukin-1β (IL-1β) and transforming growth factor-β (TGF-β). Confluent fibroblast monolayers were incubated under standard culture conditions with or without cytokines. C1qRC function was measured by attachment assays. IL-1β and TGF-β increased fibroblast adhesion to C1q to 146% and 131% of controls, respectively. Cytokine enhancement of HGF adhesion was concentration-dependent, saturable (20 ng/ml IL-1β; 1 ng/ml TGF-β) and time-dependent (IL-1β 12-hr peak; TGF-β 24-hr peak). Effect of IL-1β and TGF-β on C1qRC expression was assessed by flow cytometry measurements of fluorescence intensity of cells stained with C1q and FITC anti-C1q antibody, and by binding studies with ¹²⁵l-C1q. Cells treated with cytokines displayed a two- to four-fold increased fluorescence of cell-bound C1q compared to controls. Binding studies indicated the increased fluorescence correlated with increase in number of C1qRC in both IL-1β (4.7 × 10⁶/cell) and TGF-β (3.9 × 10⁶/cell)-treated cells, compared to control (3.0 × 10⁶/cell), but had no effect on binding affinity. Rates of internalization of receptor-bound C1q were similar in cytokine-treated cells and controls. We propose from these data that IL-1β and TGF-β have the ability to upregulate C1qRC expression, and this effect contributes to increased adhesion of HGF to substrate-bound C1q. © 1993 Wiley-Liss, Inc.     

TGF-β2 increases cell-cell communication in chondrocytes via p-Smad3 signalling

Connexin 43 (Cx43)-mediated gap junction intercellular communication (GJIC) plays a crucial role in the pathology and physiology of joint tissues. Transforming growth factor-β2 (TGF-β2), one of the potent regulatory factors in chondrocytes, plays a key role in the regulation of cell cycle and development of joint diseases. However, it is still unknown how TGF-β2 mediates GJIC in chondrocytes. The aim of this study was to explore the potential mechanism by which TGF-β2 regulates GJIC in chondrocytes. CCK-8 assays and scratch assays were performed to define the role of TGF-β2 on cell proliferation and migration. The scrape loading/dye transfer assay and scanning electron microscopy (SEM) were used to verify the effect of TGF-β2 on GJIC between chondrocytes. qPCR was performed to analyse the expression of genes in the gap junction protein family in chondrocytes. The expression of the Cx43 protein and phosphorylated Smad3 (p-Smad3) was evaluated by western blot assay. Immunofluorescence staining was used to explore p-Smad3 signalling pathway activation and Cx43 distribution. From these experiments, we found that the Cx43 protein was the most highly expressed member of the gap junction protein family in chondrocytes. We also found that TGF-β2 facilitated cell-to-cell communication in chondrocytes by upregulating Cx43 expression in chondrocytes. Finally, we found that TGF-β2 activated Smad3 signalling and promoted the nuclear aggregation of p-Smad3. Inhibition experiments by SIS3 also confirmed that TGF-β2-mediated GJIC through p-Smad3 signalling. For the first time, this study confirmed that TGF-β2 could regulate the formation of Cx43-mediated GJIC in chondrocytes via the canonical p-Smad3 signalling pathway.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.     

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) alter connexin 43 phosphorylation in MC3T3-E1 Cells

Background  

Bone morphogenetic proteins (BMPs) and transforming growth factor-βs (TGF-βs) are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC) in MC3T3-E1 cells. Connexin 43 (Cx43) has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC.     

BMP-7/TGF-β1 signalling in myoblasts: components involved in signalling and BMP-7-dependent blockage of TGF-β-mediated CTGF expression

We and others have recently described the antagonistic role of Bone morphogenetic protein-7 (BMP-7) in TGF-β signalling and myogenic differentiation. To specify the underlying mechanism(s), we here analysed the expression and function of the individual components mediating TGF-β1 and BMP-7 responses. We found that BMP-7 at a concentration of 25 ng/ml induces signalling exclusively via ALK2 and ALK3 leading to the activation of Smad1 and Smad5 and subsequent expression of Id proteins. In contrast, low doses of TGF-β1 (0.1 ng/ml) lead to an exclusive activation of ALK5 and phosphorylation of Smad2 and Smad3 that regulate specific target genes including connective tissue growth factor (CTGF). CTGF is rapidly induced by TGF-β1 already 1h after stimulation and reduced by BMP-7 application. Smad1/Smad5 or Id1/2 overexpression reduced the TGF-β1-mediated expression of CTGF. However, although siRNA-mediated knock down of Alk2/3 or Smad1/5 counteracts the BMP-7 effect on basal CTGF expression there was no consistent reversion of the observed BMP-7 effect on TGF-β1-mediated CTGF expression. Moreover, ALK5 inhibition using the SB431542 inhibitor significantly affected CTGF expression only at later time points whereas ERK1/2 inhibition completely abrogated CTGF expression. These findings point towards a regulatory role of BMP-7 that relies on modulation of Mitogen-activated protein kinases rather than mechanisms that are exclusively driven by differential Smad activation.     

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34⁺ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34⁺ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5–50 ng/ml had not effect on the expansion of CD34⁺ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34⁺ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.     

Cell-associated activation of latent transforming growth factor-β by calpain

Transforming growth factor-β (TGF-β) is normally secreted in a latent form, and plasmin-mediated proteolytic cleavage of latency-associated peptide (LAP), a component of latent TGF-β complex that makes the complex inactive, activates latent TGF-β. In the present study, we investigated the possible involvement of calpain, one of the cysteine proteases, in the activation of latent TGF-β. When recombinant latent TGF-β was incubated with calpain (1–10 u/ml) in a test tube, calpain cleaved LAP and released mature TGF-β from the latent complex. When calpain was applied to cultured bovine capillary endothelial (BCE) cells, a low concentration of calpain (0.05–0.1 u/ml) inhibited the migration and proliferation of the cells, and these inhibitory effects were abrogated by anti-TGF-β antibody as well as by calpain inhibitor peptide, but not by α₂-antiplasmin, a specific inhibitor of plasmin. Active TGF-β was detected in the conditioned medium of BCE cells collected in the presence of calpain. Chemical cross-linking of ¹²⁵I-calpain to BCE cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that calpain bound to the cell surface through chondroitinase ABC-sensitive proteoglycan. In addition, treatment of the BCE cells with chondroitinase ABC abrogated the inhibitory effect of calpain on the migration of these cells. Our data thus suggest that calpain is able to activate latent TGF-β through a mechanism independent of plasmin. This activation is efficient in the presence of cells, and calpain binds to the cell surface via proteoglycan and activates latent TGF-β, which is targeted to the same surface. J. Cell. Physiol. 174:186–193, 1998. © 1998 Wiley-Liss, Inc.     

Differential expression and biological activity of retinoic acid–induced TGFβ isoforms in embryonic palate mesenchymal cells

The effect of retinoic acid (RA) on TGF-β mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-β bioassay in association with TGF-β isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-β protein released into the culture medium. RA had little or no effect on TGF-β1 mRNA expression and protein production. In contrast, RA increased TGF-β2 and β3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-β released was increased relative to the total increase in TGF-β released, suggesting that RA treatment stimulated activation of latent TGF-β. RA also increased TGF-β2 mRNA expression; we have previously shown that RA upregulates TGF-β3 mRNA in these cells. RA and TGF-β individually inhibited ³H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit ³H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-β2 or β3-specific neutralizing antibodies partially relieved the inhibitory effect on ³H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-β. Simultaneous treatment with RA and TGF-β also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-β treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-β in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-β in retinoid-induced cleft palate. J. Cell. Physiol. 177:36–46, 1998. © 1998 Wiley-Liss, Inc.     

CTGF facilitates cell‐cell communication in chondrocytes via PI3K/Akt signalling pathway

PurposesGap junction intercellular communication (GJIC) is essential for articular cartilage to respond appropriately to physical or biological stimuli and maintain homeostasis. Connective tissue growth factor (CTGF), identified as an endochondral ossification genetic factor, plays a vital role in cell proliferation, migration and adhesion. However, how CTGF regulates GJIC in chondrocytes is still unknown. This study aims to explore the effects of CTGF on GJIC in chondrocytes and its potential biomechanism.Materials and methodsqPCR was performed to determine the expression of gene profile in the CCN family in chondrocytes. After CTGF treatment, CCK‐8 assay and scratch assay were performed to explore cell proliferation and migration. A scrape loading/dye transfer assay was adopted to visualize GJIC in living chondrocytes. Western blot analysis was done to detect the expression of Cx43 and PI3K/Akt signalling. Immunofluorescence staining was used to show protein distribution. siRNA targeting CTGF was used to detect the influence on cell‐cell communication.ResultsThe CTGF (CCN2) was shown to be the highest expressed member of the CCN family in chondrocytes. CTGF facilitated functional gap junction intercellular communication in chondrocytes through up‐regulation of Cx43 expressions. CTGF activated PI3K/Akt signalling to promote Akt phosphorylation and translocation. Suppressing CTGF also reduced the expression of Cx43. The inhibition of PI3K/Akt signalling decreased the expressions of Cx43 and thus impaired gap junction intercellular communication enhanced by CTGF.ConclusionsFor the first time, we provide evidence to show CTGF facilitates cell communication in chondrocytes via PI3K/Akt signalling pathway.     

ELF magnetic field inhibits gap junctional intercellular communication and induces hyperphosphorylation of connexin43 in NIH3T3 cells.

The effects of extremely low frequency (ELF) magnetic field on gap junctional intercellular communication (GJIC), protein levels, and phosphorylation of connexin43 (Cx43) were studied in NIH3T3 cells. The suppression of GJIC by 24 h, 50 Hz, 0.8 mT ELF magnetic field, 2 h, 3 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or ELF combined with TPA treatment was confirmed by the fluorescence recovery after photobleaching (FRAP) analysis with a confocal microscope. The results showed that ELF or TPA exposure induced 50-60% inhibition of GJIC (P < 0.01). ELF combined with TPA enhanced the inhibition of GJIC. Western blot analysis using Cx43 specific antibodies showed obviously decreasing non phosphorylated Cx43 (P(0)) induced by ELF and/or TPA exposure. On the other hand, cells treated with ELF and/or TPA displayed a hyperphosphorylated Cx43 band (P(3)). However, there was no obvious changes in the level of Cx43 protein. The results implied that the P(3) band appeared to result from phosphorylation of P(0). But it remains possible that upon the ELF exposure P(0) is converted to P(1), P(2) or both and that P(3) is formed from P(1) or P(2) resulting in the observed hyperphosphorylation pattern. From the present study, we conclude that ELF magnetic field inhibits GJIC and the main mechanism is the hyperphosphorylation of Cx43.     

TGF-β receptors are diminished after retinoid exposure in rat liver epithelial cells

When rat liver epithelial cells were exposed to retinoic acid or retinol for 24 hr, the levels of transforming growth factor-β (TGF-β) receptors were reduced in a dose-dependent way. The decrease appeared after 12 hr of incubation with the retinoids and binding levels remained low until 24 hr after the removal of the molecules. Retinoid treatment induced a fourfold enhancement of transglutaminase (TGase) activity in the cell membranes, and cystamine, an inhibitor of TGase, prevented the decrease of the receptors. Neutralization of TGF-β by a monoclonal antibody did not suppress the decrease of the binding levels, indicating that decreased TGF-β binding capacity was not due merely to the internalization of ligand-bound receptors promoted by a stimulation of TGF-β synthesis. Thus, retinoid treatment resulted in an intense disappearance of the functional receptors from the membranes that seemed to be mediated by increased TGase activity. This phenomenon can represent a strong signal attenuation for TGF-β following retinoid exposure. © 1996 Wiley-Liss, Inc.     

Propagation of Mechanically Induced Intercellular Calcium Waves via Gap Junctions and ATP Receptors in Rat Liver Epithelial Cells

Mechanical stimulation was used to initiate Ca²⁺waves in rat liver epithelial cells in order to ascertain the degree to which gap junctional intercellular communication (GJIC) is involved in communication of Ca²⁺to adjacent cells and to assess alternative Ca²⁺signaling pathways that may be present between these cells. In both WB-F344 cells, which show a high degree of GJIC, and WB-aB1 cells, which are GJIC deficient, mechanical stimulation of a single cell induced a Ca²⁺wave which propagated away from the point of stimulation, across cell borders, to neighboring cells directly or indirectly in contact with the stimulated cell. In addition, the Ca²⁺wave was transmitted to nearby isolated cells that exhibited no direct or indirect contact with the stimulated cell. Treatment of cells with 18β-glycyrrhetinic acid, a compound that has been shown to block GJIC, did not significantly affect propagation of the Ca²⁺wave. In contrast, treatment with suramin, a P₂-purinergic receptor inhibitor, significantly reduced both the rate and the extent of Ca²⁺wave propagation in WB-F344 cells and completely blocked its propagation in WB-aB1 cells. Cotreatment with suramin and glycyrrhetinic acid was found to completely block the mechanically induced Ca²⁺wave in both cell lines. These studies indicate that mechanically induced cell injury in rat liver epithelial cells initiates signaling through at least two pathways, involving intercellular communication via gap junctions and extracellular communication via ATP activation of purinergic receptors.