Anchorage-independent growth of primary rat embryo cells is induced by platelet-derived growth factor and inhibited by type-beta transforming growth factor

Several growth factors implicated in the process of cellular transformation were tested for their ability to induce anchorage-independent (AI) growth of primary rat embryo (RE) cells. Our results show that in the presence of 10% calf serum, platelet-derived growth factor (PDGF), 1-30 ng/ml, has the strongest effect of all growth factors tested on AI growth. Type-beta transforming growth factor (TGF-beta), by itself, does not stimulate AI growth, and it inhibits the PDGF-induced colony formation in a dose-dependent manner (ED50 approximately 0.03 ng/ml). Qualitatively similar responses are obtained by using an established line of fibroblasts, NIH 3T3 cells; the principal difference between the response of the primary cells and the established cell line is in colony-forming efficiency in soft agar culture (15% and 90%, respectively, for growth of colonies greater than 1,500 micron2 diameter in the presence of 10 ng/ml PDGF). Since AI growth has been shown to correlate well with tumorigenicity in vivo, our results suggest that the transforming potential of PDGF in an appropriate responsive cell can be controlled not only through its interaction with its own receptor, but also by the presence of inhibitory factors such as TGF-beta.     

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation.     

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.     

Induction and autocrine receptor binding of transforming growth factor-beta 2 during terminal differentiation of primary mouse keratinocytes

Primary cultures of mouse keratinocytes maintain a basal cell phenotype in 0.05 mM Ca2+ medium, while culture in 1.4 mM Ca2+ results in terminal differentiation and inhibition of DNA synthesis. Induction of differentiation by Ca2+ results in a 10- to 20-fold increase in the expression of transforming growth factor-beta 2 (TGF-beta 2) mRNA and peptide, but a decrease in the expression of TGF-beta 1. In contrast, binding and cross-linking analyses show that the number of available surface 80 kilodalton (kDa) and 65 kDa TGF-beta receptor types decrease during differentiation. However, a mild acid wash significantly increases the number of available receptor sites on the differentiated keratinocytes, indicating that the TGF-beta receptors are unavailable for binding due to masking by endogenous ligand. A significant level of TGF-beta 2 secretion and receptor binding occur before the decrease in DNA synthesis, suggesting that the inhibition of DNA synthesis associated with differentiation of keratinocytes is mediated through the production and autocrine action of TGF-beta 2.     

Ras-independent activation of the Raf/MEK/ERK pathway upon calcium-induced differentiation of keratinocytes

MAPKs are crucially involved in the regulation of growth and differentiation of a variety of cells. To elucidate the role of MAPKs in keratinocyte differentiation, activation of ERK, JNK, and p38 in response to stimulation with extracellular calcium was analyzed. We provide evidence that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway. Stimulation of keratinocytes with extracellular calcium resulted in activation of Raf isozymes and their downstream effector ERK within 10-15 min, but did not increase JNK or p38 activity. Calcium-induced ERK activation differed in kinetics from mitogenic ERK activation by epidermal growth factor and could be modulated by alterations of intracellular calcium levels. Interestingly, calcium stimulation led to down-regulation of Ras activity at the same time that ERK activation was initiated. Expression of a dominant-negative mutant of Ras also did not significantly impair calcium-induced ERK activation, indicating that calcium-mediated ERK activation does not require active Ras. Despite the transient nature of ERK activation, calcium-induced expression of the cyclin-dependent kinase inhibitor p21/Cip1 and the differentiation marker involucrin was sensitive to MEK inhibition, which suggests a role for the Raf/MEK/ERK pathway in early stages of keratinocyte differentiation.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

Activation of epidermal growth factor receptor promotes late terminal differentiation of cell-matrix interaction-disrupted keratinocytes

The biological effects of epidermal growth factor receptor (EGFR) activation may differ between epidermal suprabasal and basal keratinocytes, since growth factors are mitogenic in adherent cells only in the presence of cell-extracellular matrix (ECM) interaction. To investigate biological effects of EGFR activation on keratinocytes without cell-ECM interaction, we cultured normal human keratinocytes on polyhydroxyethylmethacrylate-coated plates, which disrupt cell-ECM but not cell-cell interaction. The cells initially expressed keratin 10 (K10) and then profilaggrin, mimicking sequential differentiation of epidermal suprabasal keratinocytes. The addition of EGF or transforming growth factor-alpha promoted late terminal differentiation (profilaggrin expression, type 1 transglutaminase expression and activity, and cornified envelope formation) of the suspended keratinocytes, while suppressing K10 expression, an early differentiation marker. These effects were attenuated by EGFR tyrosine kinase inhibitor PD153035 or an anti-EGFR monoclonal antibody, whereas protein kinase C inhibitors H7 and bisindolylmaleimide I or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059 abolished profilaggrin up-regulation but not K10 suppression. Since the antidifferentiative role of EGFR on cell-ECM interaction-conserved keratinocytes has been well documented, our results indicate that the biological effects of EGFR on keratinocytes are influenced by cell-ECM interaction and suggest that EGFR activation promotes rather than inhibits the terminal differentiation of suprabasal epidermal keratinocytes.     

Growth arrest induced by transforming growth factor beta 1 is accompanied by protein phosphatase activation in human keratinocytes.

Protein phosphorylation and dephosphorylation are involved in regulation of cell growth. We tested the hypothesis that the growth inhibitory effect of transforming growth factor beta 1 (TGF-beta 1) involves activation of protein phosphatases. Exposure of human keratinocytes in culture to 400 pM TGF-beta 1 for 48 h led to 80% inhibition of DNA synthesis as measured by nuclear labeling. Incubation of cultured keratinocytes with 400 pM TGF-beta 1 rapidly activated (within 30 min) protein serine/threonine phosphatase, measured using phosphorylase as a substrate. Based on several criteria, including neutralization of activity with specific antibodies and inhibitor-2, TGF-beta 1-activated phosphorylase phosphatase was identified as protein phosphatase 1. TGF-beta 1 did not have rapid effects on protein serine/threonine phosphatase activity (type 2A) measured with histone phosphorylated by protein kinase C or on protein tyrosine phosphatase activity. However, protein tyrosine phosphatase was activated at 48 h, coincident with growth arrest. Differentiation, induced by the combination of TGF-beta 1 plus calcium or by serum, was not accompanied by further serine/threonine or tyrosine phosphatase activation. We conclude that induction of growth arrest in keratinocytes by TGF-beta 1 involves acute activation of protein phosphatase 1, while activation of protein tyrosine phosphatase may represent an additional mechanism for maintaining cells in a growth-arrested state.     

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.     

Identification of a membrane-associated receptor for transforming growth factor type E

Abstract

We have identified the receptor for epithelial type transforming growth factor (TGFe). TGFe, a member of the epithelin/granulin family of proteins, is present primarily in tissues of epithelial origin. It is a powerful mitogen for epithelial and fibroblastic cells. TGFe, iodinated using an immobilized glucose oxidase-lactoperoxidase method, was chemically crosslinked to receptors on membranes isolated from SW-13 adrenal carcinoma cells by the crosslinker disuccinimidyl suberate (DSS). The receptor appears to be a protein which migrates at an apparent molecular weight of approximately 170-175 kDa under reducing and nonreducing conditions in SDS-polyacrylamide gels.     

Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta

Cultured TA1 adipocytes treated with tumor necrosis factor alpha (TNF) lose intracytoplasmic lipid and, over a period of days, come to resemble their predifferentiated progenitors (preadipocytes). To examine the extent to which this phenotypic reversion represents a return to a less differentiated cell, we examined three major characteristics that distinguish preadipocytes from adipocytes: (a) pattern of gene expression; (b) hormonal requirement for accelerated adipogenesis; and (c) pattern of protein synthesis. We found that within hours of TNF addition to adipocytes, mRNAs for genes whose expression is augmented during adipogenesis decreased to predifferentiated levels; in addition, like preadipocytes, TNF-treated adipocytes required exposure to hormones to accelerate adipogenesis. Further, the pattern of protein synthesis seen on polyacrylamide gels reverted to that seen before differentiation. Transforming growth factor-beta (TGF-beta) also caused a rapid decrease in expression of adipose genes when added to fully differentiated cells, an effect that was achieved by treatment with either TGF-beta 1 or TGF-beta 2. These effects were seen in the absence of a demonstrable proliferative response to either TNF or TGF-beta. Thus characteristics that define the "terminally" differentiated state in adipocytes are subject to modulation by environmental influences.     

Regulation of myogenic differentiation by type beta transforming growth factor

Type beta transforming growth factor (TGF beta) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation. The effects of TGF beta also are cell-type specific and appear to be modulated by other growth factors. In the present study, we examined the potential of TGF beta for control of myogenic differentiation. In mouse C-2 myoblasts, TGF beta inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor. Differentiation of the nonfusing muscle cell line, BC2Hl, was also inhibited by TGF beta in a dose-dependent manner (ID50 approximately 0.5 ng/ml). TGF beta was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation. Inhibition of differentiation required the continual presence of TGF beta in the culture media. Removal of TGF beta led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGF beta are highly transient and require continuous occupancy of the TGF beta receptor. Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGF beta inhibited differentiation at the level of muscle-specific mRNA accumulation. These results provide the first demonstration that TGF beta is a potent regulator of myogenic differentiation and suggest that TGF beta may play an important role in the control of tissue-specific gene expression during development.     

Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells

A hitherto unknown function for transglutaminase (TGase; R-glutaminyl- peptide: amine gamma-glutamyltransferase, EC 2.3.2.13) was found in the conversion of latent transforming growth factor-beta (LTGF-beta) to active TGF-beta by bovine aortic endothelial cells (BAECs). The cell- associated, plasmin-mediated activation of LTGF-beta to TGF-beta induced either by treatment of BAECs with retinoids or by cocultures of BAECs and bovine smooth muscle cells (BSMCs) was blocked by seven different inhibitors of TGase as well as a neutralizing antibody to bovine endothelial cell type II TGase. Control experiments indicated that TGase inhibitors and/or a neutralizing antibody to TGase did not interfere with the direct action of TGF-beta, the release of LTGF-beta from cells, or the activation of LTGF-beta by plasmin or by transient acidification. After treatment with retinoids, BAECs expressed increased levels of TGase coordinate with the generation of TGF-beta, whereas BSMCs and bovine embryonic skin fibroblasts, which did not activate LTGF-beta after treatment with retinoids, did not. Furthermore, both TGase inhibitors and a neutralizing antibody to TGase potentiated the effect of retinol in enhancing plasminogen activator (PA) levels in cultures of BAECs by suppressing the TGF-beta-mediated enhancement of PA inhibitor-1 (PAI-1) expression. These results indicate that type II TGase is a component required for cell surface, plasmin-mediated LTGF-beta activation process and that increased expression of TGase accompanies retinoid-induced activation of LTGF- beta.     

Effects of tumor promoters on the rate and commitment to terminal differentiation of subpopulations of murine keratinocytes

The effects of skin-tumor-promoting and -nonpromoting agents on the kinetics of terminal differentiation of subpopulations of keratinocytes differing in buoyant density isolated from mice (SENCAR) that are very sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion were investigated. Topical pretreatment of dorsal skin with complete (TPA), first-stage (calcium ionophore A23187) and second-stage (mezerein) tumor promoters, but not the hyperplastic agent ethylphenylpropiolate, accelerated the rate of terminal differentiation of keratinocytes with densities less than 1.074 g/cm3, but had little effect on cells with a greater density. Within 8.5 hr of TPA treatment, a period preceding mitosis, a large percentage of the most dense basal-cell keratinocytes (greater than or equal to 1.074 g/cm3) were converted to cells with a lower density, with a reduced plating efficiency and with an increased rate of differentiation, suggesting that TPA induces a subpopulation of basal cells to commit to terminal differentiation, and accelerates the rate of differentiation of committed cells.     

Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor.

FGF-2 exerts its pleiotropic effects on cell growth and differentiation by interacting with specific cell surface receptors. In addition, exogenously added FGF-2 is translocated from outside the cell to the nucleus during G1-S transition. In this study, we show that a single point mutation in FGF-2 (substitution of residue serine 117 by alanine) is sufficient to drastically reduce its mitogenic activity without affecting its differentiation properties. The FGF-2(S117A) mutant binds to and activates tyrosine kinase receptors and induces MAPK and p70S6K activation as strongly as the wild-type FGF-2. We demonstrate that this mutant enters NIH3T3 cells, is translocated to the nucleus, and is phosphorylated similar to the wild-type growth factor. This suggests that FGF-2 mitogenic activity may require, in addition to signaling through cell surface receptors and nuclear translocation, activation of nuclear targets. We have previously shown that, in vitro, FGF-2 directly stimulates the activity of the casein kinase 2 (CK2), a ubiquitous serine/threonine kinase involved in the control of cell proliferation. We report that, in vivo, FGF-2(WT) transiently interacts with CK2 and stimulates its activity in the nucleus during G1-S transition in NIH3T3 cells. In contrast, the FGF-2(S117A) mutant fails to interact with CK2. Thus, our results show that FGF-2 mitogenic and differentiation activities can be dissociated by a single point mutation and that CK2 may be a new nuclear effector involved in FGF-2 mitogenic activity.-Bailly, K., Soulet, F., Leroy, D., Amalric, F., Bouche, G. Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor (FGF-2).