Serum-stimulated phosphate uptake and initiation of fibroblast proliferation

Previous studies have shown that initiation of proliferation of density-inhibited fibroblasts by fresh serum is accompanied by a rapid increase in phosphate uptake. This increase might be a key event in the initiation of DNA synthesis. The present studies examined this possibility. Mouse 3T3, secondary chick embryo, or human diploid foreskin cultures were grown to quiescence in medium containing varying levels of serum. When proliferation of the cultures was initiated by addition of fresh serum, the changes in phosphate uptake were inversely related to the final increases in cell number. Additional experiments showed that the change in phosphate uptake following serum addition was determined by the level of phosphate uptake prior to serum addition. Addition of dexamethasone to quiescent 3T3 cultures caused them to proliferate but did not increase phosphate uptake. Similarly, trypsin or insulin stimulated proliferation of quiescent secondary chick embryo cultures, but caused little or no change in phosphate uptake. Quiescent 3T3 cultures switched to medium containing fresh serum and reduced levels of phosphate showed a decrease in both phosphate uptake and intracellular phosphate pool size. Cell proliferation in these cultures, however, was stimulated to the same degree as cultures switched to medium containing fresh serum and the normal amount of phosphate. In addition, quiescent secondary chick embryo cultures switched to medium containing fresh serum and no phosphate showed a decrease in the intracellular phosphate pool size. Thymidine incorporation and final cell number in these cultures, however, was stimulated to the same or higher degree than in cultures switched to medium containing fresh serum and the normal amount of phosphate. These results demonstrate that the rapid increase in phosphate uptake following addition of fresh serum to quiescent fibroblasts is not a necessary event for the initiation of proliferation.     

Genetic control of variation in human gingival fibroblast proliferation rate

Summary Logarithmic proliferation rate (Days 1 to 6) of gingival fibroblasts derived from 15 pairs of monozygotic (MZ) and 9 pairs of dizygotic (DZ) human twins was compared under optimal and suboptimal growth conditions. Cell proliferation rates exhibited considerable variability among strains. For Caucasian donors (13 MZ, 6 DZ pairs) DZ twins demonstrated significantly greater (P<0.01) within-pair variance in cell proliferation rate compared to MZ twins when evaluated under optimal growth conditions. Heritability analysis indicated strong genetic control of proliferation rate of human gingival fibroblasts (HGF) under optimal growth conditions (1.0±0.67), whereas proliferation rate of HGF under suboptimal growth conditions revealed less genetic control (0.42±0.61). These findings emphasize the importance of carefully matching control and test HGF in assays dependent on cellular proliferation. This work was supported by grants DE-06671 and DE-07841 from the National Institutes of Health.     

Hexose uptake enhancing factor released from rous sarcoma cells

Conditioned media from Rous sarcoma virus transformed chicken embryo fibroblasts stimulate the uptake of 2-deoxyglucose in normal chicken fibroblasts. The factor responsible for this effect, which is also shed in very low amount by non-transformed fibroblasts, is destroyed by trypsin and not linked to the protease and plasminogen activator activities present in the media. Its apparent molecular weight, determined by gel filtration, is about 20.000 daltons. The factor released by transformed cells might be related to the monomeric form of a family of glucose binding and transport proteins recently reported by Lee and Lipmann ('78) to be detached by detergents from normal and transformed cells.     

Selective control of fibroblast proliferation and its effect on cardiac muscle differentiation in vitro

The stability of the differentiated state of cardiac myocytes in vitro was examined under culture conditions which selectively stimulated or inhibited proliferation of fibroblasts. Regulation of fibroblast proliferation in cultures of myocardial cells from 8-day embryonic chicks was achieved by adjustment of the glutamine (Gln) concentration in the culture medium (Ham's F-12 medium containing 2 x amino acids and 5% fetal calf serum). Myocardial cells, when plated at 80 cells/mm2 in Gln- medium, maintained a stable density of approximately 40% of the plating density for more than 30 days. When Gln was added to the medium (292 micrograms/ml) fibroblast proliferation was stimulated, and by 5-6 days after this addition cell densities had increased to confluency. The selective action of glutamine on fibroblast proliferation was determined by labeling cultures with tritiated thymidine ([3H]TdR) and scoring its incorporation into myocytes and fibroblasts by radioautography. After 2 weeks in Gln- medium, the mitotic index was 0.3% and the [3H]TdR-labeling index (1.5-hr pulse) was 6.4%. In addition, the proportion of myocytes in the population was constant at 64.2% for at least 30 days in vitro, and contractile activity was observed for up to 6 months. After 5 days of Gln replacement, the cells exhibited a labeling index of 25%, the proportion of myocytes decreased to less than 10% and contractile activity was rarely observed. Although the [3H]TdR-labeling index of fibroblasts and myocytes was nearly identical in Gln- medium, the addition of Gln produced a fivefold stimulation in the fibroblast labeling index, but did not affect myocyte proliferation or DNA synthesis. A unique phenomenon of myocyte congregation was observed only in Gln- medium which resulted in the formation of myocyte colonies from which fibroblasts were largely absent. It is suggested that this process with the resultant establishment of a functional electrical syncytium plays a significant role in the development and stabilization of myocyte differentiation in vitro.     

The role of thymidine uptake in the control of cell proliferation

Using a potent inhibitor of nucleoside transport, s(p-nitrobenzyl)-6 mercaptoinosine (NBMI), we have shown that in medium 199, which contains folate levels comparable to those in serum, the multiplication of cells of bovine or murine origin can be limited by the uptake of exogenous thymidine. Moreover, the inhibition of NBMI of the growth-promoting effect of 3T6 fibroblasts for lens epithelial indicates that the feeders are acting by providing thymidine. Our results suggest that the salvage of thymidine by cells in multicellular organisms may be important in the regulation of cell growth.     

Role of pH in fibroblast proliferation

Secondary cultures of human diploid fibroblasts were used to study the effect of pH on cellular proliferation. In nonconfluent cultures, the growth rate at pH 7.1 was similar to that at pH 7.7 regardless of serum concentration. However, the saturation density achieved at pH 7.7 at any serum concentration was always 2–4 times that achieved at pH 7.1, although the greatest differences in saturation density were observed at the higher serum levels. The results suggest that the effect of pH on saturation density is due to two factors. One, cells at pH 7.1 seem to have a greater ability to undergo contact-inhibition than at pH 7.7, independent of any serum functions; and, two, confluent cells in medium at pH 7.1 are somewhat less sensitive to growth stimulation by increasing serum concentration than are confluent cells raised in medium at pH 7.7.     

Stimulation of fibroblast proliferation by thrombospondin

Thrombospondin purified from human platelets was examined for its ability to promote proliferation of human dermal fibroblasts. The results show that thrombospondin could stimulate the incorporation of [3H]thymidine by quiescent fibroblasts in a dose-dependent manner without stimulating protein or collagen synthesis. The effect was observed even in the total absence of serum, although the degree of stimulation was substantially lower than that in the presence of 0.4% fetal calf serum, but higher than that in the presence of 4% serum. The effect was specific and not due to contaminants as demonstrated by the ability of antibodies to thrombospondin to specifically inhibit this stimulation. Three monoclonal antibodies directed at different epitopes in the thrombospondin molecule were equally effective in inhibiting this effect. This stimulation of fibroblast proliferation by thrombospondin suggests a potential role for this matrix protein in the mesenchymal cell response in tissue injury and repair.     

IQGAP1 regulates hyaluronan-mediated fibroblast motility and proliferation

IQGAP1, an essential scaffolding protein, forms a complex with the hyaluronan receptor CD44. In this study, we have examined the importance of IQGAP1 for hyaluronan-mediated fibroblast migration and proliferation. Hyaluronan induced formation of F-actin fibers and focal adhesions, which was dependent on IQGAP1. IQGAP1 was required for hyaluronan- but not for platelet-derived growth factor (PDGF)-BB-induced cell migration, and was required for both hyaluronan- and PDGF-BB-mediated fibroblast proliferation, but not for proliferation induced by 10% fetal bovine serum. Depletion of IQGAP1 suppressed hyaluronan-induced activation of Rac1 and enhanced the activation of RhoA. Taken together, these findings indicate important roles for IQGAP1 in hyaluronan-stimulated migration and proliferation of fibroblasts.     

Lymphocyte-mediated activation of fibroblast proliferation and collagen production.

Among the many activities of antigen-triggered lymphocytes may be the control of fibroblast function. Thymus-dependent lymphocytes challenged with the specific antigen, dinitrophenylated ovalbumin or tetanus toxoid produced a nondialyzable factor(s) capable of causing dermal fibroblasts to undergo DNA synthesis. These fibroblasts, which exhibit basal proliferative levels in the absence of serum, responded to the lymphocyte factor with maximal thymidine incorporation at 48 to 72 hr. In addition, these activated fibroblasts significantly increased their production of protein of both the collagenous and noncollagenous types. This increase in protein synthesis preceded maximal proliferation. Thus, the fibroplasia consisting of increased numbers of fibroblasts and of increased collagen deposition associated with chronic inflammatory diseases may be the direct consequence of a specific antigenic challenge.     

Hexose uptake by Catharanthus roseus cell suspensions is inhibited by a high medium salt content

Summary The uptake of glucose and fructose from the medium by Catharanthus roseus cell suspensions was strongly inhibited by high medium salt concentration, such as found in LS (Linsmaier and Skoog 1965) medium. After inoculation into standard LS nutrient medium with less than 5 mM hexose no uptake occurred, while in low salt medium hexose was completely depleted. At a hexose concentration of 50 mM the uptake rate was higher in low salt medium than in standard medium. The lower rate of uptake at high salt concentration was not the result of a pH or osmotic effect of the salts. Probably the affinity of the hexose carrier is affected by the ion concentration of the medium. The decrease in medium salt concentration during normal batch culture probably will have a considerable effect on hexose uptake.Abbreviations LS Linsmaier and Skoog - S sucrose - N mineral nitrogen - K K₂SO₄ - F fructose     

Role of interleukin-6 in cardiomyocyte/cardiac fibroblast interactions during myocyte hypertrophy and fibroblast proliferation

The process of cardiac hypertrophy is considered to involve two components: that of cardiac myocyte (CM) enlargement and cardiac fibroblast (CF) proliferation. The interleukin-6 (IL-6) family cytokines have been implicated in a variety of cellular and molecular interactions between myocytes and non-myocytes (NCMs), which in turn have important roles in the development of cardiac hypertrophy. In the study of these interactions, we previously detected very high levels of IL-6 in supernatants of a "dedifferentiated model" of adult ventricular CMs cultured with CFs. In the present study, we have used this in vitro coculture system to examine how IL-6 is involved in the interactions between CMs and CFs during CM hypertrophy and CF proliferation. IL-6 and its signal transducer, 130-kDa glycoprotein (gp130), were detected by immunostaining cultured CMs and CFs with anti-IL-6 or anti-gp130 antibodies. Addition of anti-IL-6 or anti-gp130 antagonist antibodies into CM/CF cocultures induced a significant decrease in expression of atrial natriuretic peptide (ANP) and beta-myosin heavy chain (beta-MHC) in CMs. The presence of IL-6 antagonist also resulted in a decrease in the surface area of 12-day-old CMs cultured with CFs or in the presence of fibroblast conditioned medium (FCM), and decreased fibroblast proliferation in CM/CF cocultures, particularly in the presence of a gp130 antagonist. The results also show that angiotensin II (AngII) is mainly secreted by CFs and induces IL-6 secretion in CMs cultured with CFs or with FCM. In addition, the effects of IL-6 on cardiomyocyte hypertrophy and fibroblast proliferation were inhibited by addition of the AT-1 receptor antagonist, losartan. These results suggest that IL-6 contributes significantly to CM hypertrophy by an autocrine pathway and to fibroblast proliferation by a paracrine pathway and that these effects could be mediated by AngII.     

硝普钠促肺成纤维细胞增殖和凋亡的作用

用MTT测定、琼脂糖凝胶电泳和流式细胞术等方法,观察了一氧化氮供体硝普钠（sodium nitroprusside,SNP）对体外培养的肺成纤维细胞增殖和凋亡以及Bcl-2、Bax和p53蛋白含量的影响。结果发现：MTT吸光度、细胞数和增殖指数（proliferation index,PI）均较对照增加;凋亡细胞数也增加,但尚不足以出现明显的“梯形”凋亡电泳条带;同时,细胞内Bcl-2蛋白下调和Bax蛋白上调;而细胞内p53蛋白含量无明显变化。结果表明,外源性NO有增强肺成纤维细胞增殖和凋亡的双重作用,但以促细胞增殖为主;此作用的分子机制与Bcl-2蛋白下调和Bax蛋白上调有关。     

Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Prolonged activation of CFs leads to cardiac fibrosis and reduced myocardial contractile function. Resveratrol (RES) exhibits a number of cardioprotective properties; however, the possibility that this compound affects CF function has not been considered. The current study tests whether RES directly influences the growth and proliferation of CFs and differentiation to the hypersecretory myofibroblast phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase activation. This inhibition by RES reduced basal proliferation and blocked ANG II-induced growth and proliferation of CFs in a concentration-dependent manner, as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively. RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with 0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK activation over basal was similar to the phosphorylation induced by 100 nM ANG II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II- and transforming growth factor-beta-induced CF differentiation to the myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin expression and stress fiber organization in CFs. This study identifies RES as an anti-fibrotic agent in the myocardium by limiting CF proliferation and differentiation, two critical steps in the pathogenesis of cardiac fibrosis.     

Suppression of fibroblast proliferation and lysyl hydroxylase activity by minoxidil

The effect of minoxidil on lysyl hydroxylase activity and proliferation of human skin fibroblasts in culture was examined. Exposure of cells to minoxidil resulted in a specific loss of lysyl hydroxylase activity, the extent of which was dependent on the concentration of minoxidil from 25 to 500 microM and the duration of the treatment from 6 to 48 h. This phenomenon was unaffected by culture conditions, i.e. ascorbic acid status, serum concentration, and cell density. Minoxidil added directly to cell extracts had no effect on lysyl hydroxylase activity, showing a requirement for intact cells. Mixing experiments with extracts of minoxidil-treated cells and controls gave additive results which rule out the possibility that a metabolite derived from minoxidil could be inhibiting the enzyme activity. The effect of minoxidil on fibroblast lysyl hydroxylase activity disappeared in the presence of cycloheximide, an inhibitor of protein synthesis. Moreover, the recovery of the enzyme activity that occurred after removal of minoxidil from the culture medium could be prevented by actinomycin D, an inhibitor of RNA synthesis. These results indicate that minoxidil may inhibit the synthesis of lysyl hydroxylase in the cell. In addition to suppressing fibroblast lysyl hydroxylase activity, minoxidil caused inhibition of cell growth within 48 h in a manner dependent on the concentration from 10 to 1000 microM, the latter resulting in almost complete cessation of cell proliferation. This effect was not accompanied by cytotoxicity as judged by the criteria of dye exclusion, plating efficiency, growth recovery, and protein synthesis. The inhibition of fibroblast proliferation by minoxidil appeared to be related to its ability to inhibit DNA synthesis measured by incorporation of tritiated thymidine into acid-precipitable material.     

Hexose sugar transport activity in a mouse fibroblast cell line temperature sensitive for expression of the transformed phenotype

A temperature-sensitive mouse fibroblast cell line was used to examine the relationship between hexose sugar uptake rates and the control of cell growth. The cell line used (ts-H6-15) is a derivative of SV-3T3 cells, exhibiting a transformed phenotype at 32°C and a normal phenotype at 39°C. For cells actively growing at either temperature, a marked decrease in the rate of 3-0-methyl-D-glucose (3-0-MeG) transport is observed as cell population density increases. At all cell population densities tested, 3-0-MeG transport rates (at a common assay temperature) were greater in H6-15 cells grown at 32°C than at 39°C, with the enhancement being maximal at the lowest cell densities. The effect of low serum-arrest on H6-15 cells revealed that cells growing at 39°C arrest in G1, while cells at 32°C stop more randomly throughout their cycle. Under conditions of low serum-arrest the rate of 3-0-MeG transport remained as high as in actively growing cells at both 32°C and 39°C. However, 2-deoxyglucose uptake rates were growth state-dependent at 39°C, indicating perhaps metabolic as well as membrane-level control of sugar accumulation. These results further demonstrate that rates of hexose sugar transport by themselves are not always absolutely correlated with rates of cell proliferation and, thus, may not be reliable predictors of cell growth potential.     

Hexose kinases of avocado

The subcellular location and properties of enzymes concerned with the phosphorylation of glucose and fructose in avocado (Persea americana Mill. cv. Hass) have been studied. A substantial amount of glucose-phosphorylating activity was particulate and fractionation of extracts by sucrose density gradient centrifugation indicated that most of this activity was associated with the mitochondria. Three hexose-phosphorylating enzymes were resolved by DEAE-cellulose chromatography of the cytosolic fraction. These were a hexokinase (EC 2.7.1.1), which had strong preference for glucose as substrate, and two specific fructokinases (EC 2.7.1.4). ATP was the preferred phosphoryl donor for the hexose kinases of avocado.