Participation of phospholipase D and alpha/beta-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts

We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAbetaC2-1 peptide (Cl8 HAbetaC2-1 cells), with a sequence (betaC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of alpha-PKC in PLD activation is further underscored by co-immunoprecipitation of alpha-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAbetaC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAbetaC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a alphaPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.     

Platelet-derived growth factor stimulates non-mitochondrial Ca2+ uptake and inhibits mitogen-induced Ca2+ signaling in Swiss 3T3 fibroblasts

Changes in intracellular free Ca2+ concentration [( Ca2+]i) were used to study the interaction between mitogens in Swiss 3T3 fibroblasts. Platelet-derived growth factor (PDGF) produced an increase in [Ca2+]i and markedly decreased the increases in [Ca2+]i caused by subsequent addition of bradykinin and vasopressin. If the order of the additions was reversed the [Ca2+]i response to PDGF was not inhibited by bradykinin or vasopressin. Inhibition of protein kinase C by staurosporine or chronic treatment of the cells with phorbol 12-myristate 13-acetate prevented the inhibitory effect of PDGF on the [Ca2+]i response to vasopressin but not bradykinin. PDGF did not decrease the receptor binding of bradykinin and produced only a small decrease in the receptor binding of vasopressin. PDGF decreased the rise in [Ca2+]i caused by the Ca2+ ionophores 4-bromo-A23187 and ionomycin and by a membrane perturbing ether lipid, 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, both in the presence and absence of external Ca2+. There was no change in cell 45Ca2+ influx caused by PDGF, vasopressin, or bradykinin. 45Ca2+ efflux from cells exposed to PDGF and vasopressin mirrored the changes in [Ca2+]i caused by the agents, that is, PDGF added after vasopressin produced a further increase in 45Ca2+ efflux but vasopressin did not increase 45Ca2+ efflux after PDGF. PDGF but not vasopressin caused an increase in the uptake of 45Ca2+ into an inositol 1,4,5-trisphosphate-insensitive non-mitochondrial store in permeabilized cells. The results suggest that the decreased [Ca2+]i response to mitogens after PDGF represents an action of PDGF at a point beyond the release of intracellular Ca2+ and the influx of external Ca2+, caused by an increase in the rate of removal of cytoplasmic free Ca2+. This increased removal of cytoplasmic Ca2+ by PDGF is not due to the increased export of Ca2+ from the cell but results from increased Ca2+ uptake into non-mitochondrial stores.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Ribozyme-mediated perlecan knockdown impairs chondrogenic differentiation of C3H10T1/2 fibroblasts

Perlecan (Pln) is an abundant heparan sulfate (HS) proteoglycan in the pericellular matrix of developing cartilage, and its absence dramatically disrupts endochondral bone formation. This study examined two previously unexamined aspects of the function of Pln in mesenchymal chondrogenesis in vitro. Using the well-established high-density micromass model of chondrogenic differentiation, we first examined the requirement for endogenous Pln synthesis and secretion through the use of Pln-targeted ribozymes in murine C3H10T1/2 embryonic fibroblasts. Second, we examined the ability of the unique N-terminal, HS-bearing Pln domain I (PlnDI) to synergize with exogenous bone morphogenetic protein-2 (BMP-2) to support later stage chondrogenic maturation of cellular condensations. The results provide clear evidence that the function of Pln in late stage chondrogenesis requires Pln biosynthesis and secretion, because 60%-70% reductions in Pln greatly diminish chondrogenic marker expression in micromass culture. Additionally, these data support the idea that while early chondrocyte differentiation can be supported by exogenous HS-decorated PlnDI, efficient late stage PlnDI-supported chondrogenesis requires both BMP-2 and Pln biosynthesis.     

Modulation by retinyl acetate of microfilament bundle formation in C3H/10T1/2 cells

Retinyl acetate has been previously shown to inhibit carcinogen-induced neoplastic transformation in 10T1/2 cells and to accentuate many aspects of the nontransformed phenotype. Scanning electron microscopy of logarithmic phase 10T1/2 cells treated for 3 days with 0.3 micrograms/ml retinyl acetate revealed that this treatment caused extensive flattening of cells to the plastic substrate. In contrast the tumor promoter tetradecanoyl phorbol acetate, which antagonizes the antineoplastic activity of retinyl acetate, caused cell rounding and completely inhibited the action of retinyl acetate on cell morphology. During this same time course, the formation of microfilament bundles was also found to be modulated by retinyl acetate. Transmission electron micrographs of unsectioned peripheral regions of flattened cells showed that while the unit density of microfilament bundles was not influenced, the thickness of bundles, particularly those with a diameter of 100 nm or more, was increased by retinyl acetate. Tetradecanoyl phorbol acetate had little effect on microfilament bundle diameters but did partially antagonize the action of retinyl acetate. To determine if this increase was associated with an increase in total actin/cell, total cell proteins, and proteins not extractable by glycerol-triton extraction, were subjected to sodium dodecylsulfate/ polyacrylamide gel electro-phoresis. It was found that while total cellular actin was not increased by retinyl acetate, the proportion of nonextractable actin (which includes microfilament bundles) increased from 65% to 88% of total actin. This increase was not inhibited by inhibitors of protein or RNA synthesis. These studies again demonstrate that retinyl acetate accentuates the nontransformed phenotype of 10T1/2 cells; it is hypothesized that these actions are related to the antineoplastic activity of retinoids.     

Molecular phenotyping of non-transformed and chemically transformed C3H10T1/2 mouse fibroblasts

A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.     

DNA binding of aflatoxin B1 by co-oxygenation in mouse embryo fibroblasts C3H/10T1/2

The mechanism of activation of aflatoxin B1 to ultimate metabolites capable of DNA binding was investigated in mouse embryo fibroblasts C3H/10T1/2. The contribution of co-oxygenation reactions which are coupled to arachidonic acid metabolism was assessed by the use of inhibitors of prostaglandin endoperoxide synthetase and lipoxygenase. Indomethacin and 5,8,11,14-icosatetraynoic acid inhibited AFB1-binding to maximally 60%. The antioxidant glutathione was also inhibitory while CuZn superoxide dismutase had no effect or slightly stimulated binding at high concentrations. These results indicate that co-oxygenation plays a major role in AFB1-metabolism in 10T1/2 cells. The observation that the phospholipase A2 inhibitor p-bromophenacylbromide diminished AFB1-DNA binding supports the notion that AFB1, because it is membrane-active, may enhance its own co-oxidative metabolism by stimulating the arachidonic acid cascade.     

The anti-apoptotic protein Mcl-1 inhibits mitochondrial Ca2+ signals

Apoptosis contributes to the regulation of cell growth and regeneration and to the development of neoplasia. Mcl-1 is an anti-apoptotic protein that is particularly important for the development of hematological and biliary malignancies, but the mechanism of action of Mcl-1 is unknown. A number of pro- and anti-apoptotic proteins exhibit their effects by modulating Ca2+ signals, so we examined the effects of Mcl-1 on components of the Ca2+ signaling pathway that are known to regulate apoptosis. Expression of Mcl-1 did not affect expression of the inositol 1,4,5-trisphosphate receptor or the size of endoplasmic reticulum Ca2+ stores. However, mitochondrial Ca2+ signals induced by either Ca2+ agonists or apoptotic stimuli were decreased in cells overexpressing Mcl-1 and increased in cells in which Mcl-1 expression was inhibited. These findings provide evidence that Mcl-1 directly inhibits Ca2+ signals within mitochondria, which may provide a novel mechanism to inhibit apoptosis and thereby promote neoplasia.     

Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells

Oscillatory mechanical stimulation at relatively high frequencies (0.1 Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01 Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01 Hz frequency for 6h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARγ after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages.     

Lactate-mediated changes in growth morphology and transformation frequency of irradiated C3H 10T1/2 cells

Treatment of mammalian cells with lactate or inhibitors of glycolysis alters their radiation response, particularly in the low dose region of the dose response curve. The occurrence of both high lactate levels and high glycolytic metabolism in tumours is well known and therefore the effect of lactate on a cell line sensitive to radiation induced transformation was examined using a single exposure to Cobalt 60 gamma rays as the carcinogen challenge. The results indicate that cells treated with 5mM lactate before irradiation exhibit changes in morphology and growth rate and that the transformation frequency is increased by three to ten fold following 24 hours lactate treatment just prior to irradiation. Examination of radiation survival curves showed a positive correlation between transformation frequency and size of the shoulder, but increasing transformation frequency was associated with a decrease in Do. A mechanism involving altered Redox potential in lactate treated cells is suggested. The results are discussed in terms of their possible significance for radiotherapy.     

BMP2具有诱导C3H10T1/2细胞成脂肪分化的能力

探讨骨形态发生蛋白2（BMP2）诱导鼠胚胎间充质干细胞C3H10T1/2成脂肪分化能力,为临床脂肪代谢疾病的治疗提供理论基础．培养多潜能的间充质干细胞C3H10T1/2,用20 μg/ml BMP2对其诱导一定时间后,RT-PCR检测是否存在BMP信号通路中关键分子BMP受体BMPR I, BMPR Ⅱ及Smad 1/5/8的表达.Western印迹检测Smad 蛋白及MAPK 信号通路中p38磷酸化水平变化,QRT PCR检测成脂肪标志基因aP2以及成脂肪相关转录因子PPARγ,C/EBPα,C/EBPβ表达水平,同时用油红O染色,观测C3H10T1/2细胞成脂肪分化情况．经BMP2诱导后,C3H10T1/2细胞成脂肪分化标志(油红O染色)显著增加,Smad 蛋白及p38磷酸化水平有所上升,同时成脂肪标志基因aP2以及成脂肪相关转录因子PPARγ,C/EBPα,C/EBPβ表达水平各有一定程度提高．BMP2具有诱导C3H10T1/2细胞成脂肪分化能力,其成脂肪分化呈现对BMP2作用的时间依赖性．     

Interrelationship between growth factor-induced activation of the Na+/H(+)-antiporter and mobilization of intracellular Ca2+ in NIH3T3-fibroblasts

Addition of serum growth factors or bombesin to quiescent NIH3T3-fibroblasts leads to a simultaneous mobilization of intracellular Ca2+ and an increase in cytosolic pH which is inhibitable by dimethylamiloride. The mobilization of intracellular Ca2+ is a pH-dependent process with an optimum at pH 7.1. In quiescent cells with a pHi greater than or equal to 6.8, inhibition of the Na+/H(+)-antiporter by dimethylamiloride or reduction of extracellular Na+ attenuates the growth factor induced Ca2(+)-response. It is concluded that the growth factor induced activation of the Na+/H(+)-antiporter facilitates the mobilization of Ca2+ by shifting the internal pH towards the optimum for the Ca2(+)-release.     

Neutron-energy-dependent oncogenic transformation of C3H 10T1/2 mouse cells

The relative biological effectiveness (RBE) of a range of neutron energies relative to 250-kVp X rays has been determined for oncogenic transformation and cell survival in the mouse C3H 10T 1/2 cell line. Monoenergetic neutrons at 0.23, 0.35, 0.45, 0.70, 0.96, 1.96, 5.90, and 13.7 MeV were generated at the Radiological Research Accelerator Facility of the Radiological Research Laboratories, Columbia University, and were used to irradiate asynchronous cells at low absorbed doses from 0.05 to 1.47 Gy. X irradiations covered the range 0.5 to 8 Gy. Over the more than 2-year period of this study, the 31 experiments provided comprehensive information, indicating minimal variability in control material, assuring the validity of comparisons over time. For both survival and transformation, a curvilinear dose response for X rays was contrasted with linear or nearly linear dose responses for the various neutron energies. RBE increased as dose decreased for both end points. Maximal RBE values for transformation ranged from 13 for cells exposed to 5.9-MeV neutrons to 35 for 0.35-MeV neutrons. This study clearly shows that over the range of neutron energies typically seen by nuclear power plant workers and individuals exposed to the atomic bombs in Japan, a wide range of RBE values needs to be considered when evaluating the neutron component of the effective dose. These results are in concordance with the recent proposals in ICRU 40 both to change upward and to vary the quality factor for neutron irradiations.     

Glutaraldehyde-fixed transformed and non-transformed cells induce contact-dependent inhibition of growth in non-transformed C3H/10T1/2 mouse fibroblasts, but not in 3-methylcholanthrene-transformed cells

C3H/10T1/2 mouse fibroblasts showed a pronounced inhibition of growth when reaching a critical cell density. The situation of high cell density could be mimicked by the addition of glutaraldehyde-fixed cells to sparsely seeded proliferating cells. Treatment of the C3H/10T1/2 cells with 3-methylcholanthrene led to a high frequency of piled up foci (118 type II and type III foci in 78 cultures). Cells of a type III focus of a treated culture were cloned. These cells grew in soft-agar and reached 10 times higher cell densities when grown in culture dishes, than did their non-transformed counterparts. Glutaraldehyde-fixed transformed cells did not differ from fixed non-transformed cells in the ability to inhibit the growth of sparsely seeded non-transformed cells. On the other hand, both the addition of fixed normal or transformed C3H/10T1/2 cells did not affect the growth rate of transformed cells. In a concept explaining the density-dependent inhibition of growth of non-transformed cells by a specific interaction of plasma membrane-localized effectors with plasma membrane-localized receptors, the present findings would indicate that the transformed cells used express active effectors but are functionally defective in the receptors or in the signal transmission.     

Radiosensitivity parameters for neoplastic transformations in C3H10T1/2 cells

We have evaluated radiosensitivity parameters for cellular transformation from published experimental data on neoplastic transformations induced in C3H10T1/2 cells by BEVALAC ions. The measured RBE values are well reproduced by a track theory calculation using sets of m-target parameters with either m = 2 or m = 3, suggesting a quadratic or cubic extrapolation to low doses of gamma rays. Using track theory one is thus able to predict transformation frequencies in those cells after an arbitrary radiation field, under known or assumed conditions of exposure, in a manner shown earlier for cellular survival. Extension of these calculations to interpret cancer incidence in vivo is also discussed.     

SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling

Background

The directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.

Results

This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.

Conclusions

Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

     

Plasma membrane hyperpolarization and [Ca2+]i increase induced by fibroblast growth factor in NIH-3T3 fibroblasts: resemblance to early signals generated by platelet-derived growth factor

The effects of different substances on [Ca2+]i and membrane potential (measured by fura-2 and bis-oxonol fluorescence techniques, respectively) were studied in wild-type and NIH-3T3 fibroblasts transfected with the cDNA encoding the human epidermal growth factor receptor. Application of partially purified PDGF or FGF induced, after a lag (0.5-1 min), a [Ca2+]i increase composed by an initial, slow peak, sustained primarily by intracellular Ca2+ release followed by a plateau, sustained by Ca2+ influx from the medium. The [Ca2+]i changes were paralleled by plasma membrane hyperpolarization mainly due to the activation of a K+ efflux, since raising the extracellular K+ concentration progressively reversed the effect of both growth factors. These responses were much slower than those induced by other agents (bradykinin, extracellular ATP, and EGF). The close resemblance between PDGF- and FGF-induced early signals (time-course and insensitivity to phorbol esters) suggests similar transmembrane signalling mechanisms at the cognate receptor.     

Thy-1 inhibits mitogen-induced Ca2+ oscillation in ras-transformed mouse fibroblasts.

Cell surface glycoprotein Thy-1 functions as a transformation suppressor in v-ras-transformed NIH/3T3 cells [Sugimoto et al., (1991) Cancer Res. 51, 99-104.]. In order to understand the mechanism of action of Thy-1, we examined the effect of Thy-1 expression on mitogen-induced Ca2+ oscillation which was correlated with v-ras-transformation [Fu et al., (1991) FEBS Lett. 281, 263-266.]. Forced expression of Thy-1 in v-ras-transformed cells inhibited mitogen-induced Ca2+ oscillation. Although v-Ras-free, Thy-1-positive NIH/3T3 cells (major population) did not show Ca2+ oscillation, whereas in Thy-1-negative NIH/3T3 cells (less than 1% of the population) Ca2+ oscillation was observed. Finally, replacement of the carboxyl-half of Thy-1 with that of CD4 abolished the inhibitory effect of Thy-1. These results suggest that Thy-1 directly or indirectly participates in the negative regulation of Ca2+ response by inhibiting Ca2+ oscillation.