Teratocarcinoma cells exhibit growth cooperativity in vitro

Malignant PC13 embryonal carcinoma (EC) cells differentiate in vitro in response to retinoic acid, giving rise to a population of benign endoderm-like cells (END), termed PC13 END. PC13 EC and PC13 END cells exhibit growth cooperativity in co-culture, whereby the EC cells stimulate END cell proliferation and the END cells can support EC cell multiplication. The EC cells' stimulatory effect operates via soluble, diffusible factors which are also active on a range of fibroblast cell lines. END cells support the multiplication of EC cells plated at low density, via a multifactorial mechanism. Contact-dependent effects can operate in the absence of END cell metabolic activity, while contact-independent effects require the continuous presence of live END cells. It was observed that there was a variation in the ability of fibroblast cell lines to act as EC cell feeders. Similar interactive events may be important during the in vivo proliferation and differentiation of teratocarcinoma cells and their embryonic counterparts.     

Production of PDGF-like growth factors by embryonal carcinoma cells and binding of PDGF to their endoderm-like differentiated cells

In this report, we demonstrate that F9 and PC-13 embryonal carcinoma (EC) cells do not bind significant amounts of platelet-derived growth factor (PDGF), whereas the endoderm-like differentiated cells derived from EC cells do. The F9-differentiated cells exhibit approximately 8300 receptors per cell, with an apparent dissociation constant of 30 pM. Two endoderm-like cell lines, PSA-5E and PYS-2, also bind PDGF and exhibit approximately 4800 and 23,500 receptors per cell, respectively. The lack of PDGF binding by the parental EC cells is consistent with their release of a factor(s) that is closely related to PDGF. This factor(s) competes with PDGF for binding to membrane receptors and is recognized by antibodies raised against PDGF. However, this factor(s) does not appear to be antigenically identical to PDGF. We also show that production of this PDGF-like factor(s) is reduced more than 90% when F9 EC cells differentiate into cells that bind PDGF. Thus, our findings indicate that EC cells release a factor(s) that should be capable of binding to their differentiated cells. This raises the possibility that PDGF, or a closely related factor, can influence cell proliferation and/or cell behavior of early embryonic cells.     

Retinoic acid abolishes the calcitonin gene-related peptide autocrine system in F9 teratocarcinoma cells

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation. J. Cell. Biochem. 64:447–457. © 1997 Wiley-Liss, Inc.     

Mitogenesis in fetal rat bone cells simultaneously exposed to type beta transforming growth factor and other growth regulators

Type beta transforming growth factor (TGF-beta) is found in large amounts in bone tissue, and is a potent mitogen for osteoblast-enriched cell cultures obtained from fetal rat parietal bone. Because other local and systemic factors may be presented to bone cells simultaneously with TGF-beta, it is important to understand the effects of this complex growth regulator in such circumstances. Unlike the effects observed in many tissue systems, TGF-beta does not invariably inhibit the mitogenic response of bone cells to other growth promoters. In contrast, other factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and type alpha tumor necrosis factor (TNF-alpha) limit the response of osteoblastic bone cells to TGF-beta. TGF-beta is a much weaker mitogen for fibroblastic cells obtained from fetal rat bone, whereas fetal bovine serum, EGF, bFGF, and TNF-alpha are more potent stimulators. In addition, TGF-beta does not significantly impair the response of the fibroblastic bone cells to the other tested agents. These findings reinforce a role of TGF-beta as an anabolic bone growth regulator, and suggest that its function may be modified by other local or systemic agents that can also affect bone cells.     

IGFBP-3 mediates TGF-beta1-induced cell growth in human airway smooth muscle cells

Both insulin-like growth factor binding protein-3 (IGFBP-3) and transforming growth factor-beta (TGF-beta) have been separately shown to have cell-specific growth-inhibiting or growth-potentiating effects. TGF-beta stimulates IGFBP-3 mRNA and peptide expression in several cell types, and TGF-beta-induced growth inhibition and apoptosis have been shown to be mediated through the induction of IGFBP-3. However, a link between the growth stimulatory effects of TGF-beta and IGFBP-3-induction has not been shown. In this study, we investigated the role of IGFBP-3 in mediating TGF-beta1-induced cell growth using human airway smooth muscle (ASM) cells as our model. TGF-beta1 (1 ng/ml) treatment induced a 10- to 20-fold increase in the levels of expression of IGFBP-3 mRNA and protein. Addition of either IGFBP-3 or TGF-beta1 to the growth medium resulted in an approximately twofold increase in cell proliferation. Coincubation of ASM cells with IGFBP-3 antisense (but not sense) oligomers as well as with an IGFBP-3 neutralizing antibody (but not with control IgG) blocked the growth induced by TGF-beta1 (P < 0.001). Several IGFBP-3-associated proteins were observed in ASM cell lysates, which may have a role in the cellular responses to IGFBP-3. These findings demonstrate that IGFBP-3 is capable of mediating the growth stimulatory effect of TGF-beta in ASM cells.     

Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth

We have previously hypothesized that the development of severe angioproliferative pulmonary hypertension is associated with not only initial endothelial cell (EC) apoptosis followed by the emergence of apoptosis-resistant proliferating EC but also with proliferation of vascular smooth muscle cells (VSMC). We have demonstrated that EC death results in the selection of an apoptosis-resistant, proliferating, and phenotypically altered EC phenotype. We postulate here that the initial apoptosis of EC induces the release of mediators that cause VSMC proliferation. We cultured EC in an artificial capillary CellMax system designed to simulate the highly efficient functions of the human capillary system. We induced apoptosis of microvascular EC using shear stress and the combined VEGF receptor (VEGFR-1 and -2) inhibitor SU-5416. Flow cytometry for the proliferation marker bromodeoxyuridine showed that serum-free medium conditioned by apoptosed EC induced proliferation of VSMC, whereas serum-free medium conditioned by nonapoptosed EC did not. We also show that medium conditioned by apoptosed EC is characterized by increased concentrations of transforming growth factor (TGF)-beta1 and VEGF compared with medium conditioned by nonapoptosed EC and that TGF-beta1 blockade prevented the proliferation of cultured VSMC. In conclusion, EC death induced by high shear stress and VEGFR blockade leads to the production of factors, in particular TGF-beta1, that activate VSMC proliferation.     

Endoderm-secreted factor stimulates growth of embryonal carcinoma stem cells

Summary Stem cells of the embryonal carcinoma cell line called H6 can be induced to differnetiate to endoderm-like cells by retinoic acid (3×10⁻⁶ M). We have detected a diffusible and stable factor which is secreted by H6 endoderm-like cells and stimulates the growth of H6 stem cells. The stimulation by the endoderm-like cells is considereably greater than that by mouse fibroblasts or H6 stem cells themselves. No reciprocal stimulation of endoderm-like cells by stem cells occurs. Part but not all of the stimulation might be due to extracellular matrix proteins or to insulin-like growth factor type 2, each of which also stimulates the growth of H6 stem cells. Insulin causes no such stimulation. This work was supported by research rant no. CA-16754 from the National Cancer Institute to J. W. L. E. L. G. was supported by an American Heart Association Medical Student Research Award. Editor's Statement This paper presents a good example of cooperativity between undifferentiated teratoma stem cells and differentiated parietal endoderm-derived countrparts in terms of growth support. It raises the interesting question of the relationship between factors produced by paprietal and visceral endoderm cells. Gordon H. Sato     

Differentiation of EC cells in vitro by the fluorescent dye Hoechst 33342

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.     

Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth

The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.     

Transforming growth factor beta is an important immunomodulatory protein for human B lymphocytes

The growth and differentiation of B cells to immunoglobulin (Ig)-secreting cells is regulated by a variety of soluble factors. This study presents data that support a role for transforming growth factor (TGF)-beta in this regulatory process. B lymphocytes were shown to have high-affinity receptors for TGF-beta that were increased fivefold to sixfold after in vitro activation. The addition of picogram quantities of TGF-beta to B cell cultures suppressed factor-dependent, interleukin 2 (IL 2) B cell proliferation and markedly suppressed factor-dependent (IL 2 or B cell differentiation factor) B cell Ig secretion. In contrast, the constitutive IgG production by an Epstein Barr virus-transformed B cell line was not modified by the presence of TGF-beta in culture. This cell line was found to lack high-affinity TGF-beta receptors. The degree of inhibition of B cell proliferation observed in in vitro cultures was found to be dependent not only on the concentration of TGF-beta added but also on the concentration of the growth stimulatory substance (IL 2) present. By increasing the IL 2 concentrations in culture, the inhibition of proliferation induced by TGF-beta could be partially overcome. In contrast, the inhibition of Ig secretion induced by TGF-beta could not be overcome by a higher concentration of stimulatory factor, demonstrating that the suppression of B cell differentiation by TGF-beta is not due solely to its effects on proliferation. Furthermore, it was demonstrated that B lymphocytes secrete TGF-beta. Unactivated tonsillar B cells had detectable amounts of TGF-beta mRNA on Northern blot analysis, and B cell activation with Staphylococcus aureus Cowan (SAC) resulted in a twofold to threefold increase in TGF-beta mRNA. Supernatants conditioned by unactivated B cells had small amounts of TGF-beta, SAC activation of the B cells resulted in a sixfold to sevenfold increase in the amount of TGF-beta present in the supernatants. Thus, B lymphocytes synthesize and secrete TGF-beta and express receptors for TGF-beta. The addition of exogenous TGF-beta to cultures of stimulated B cells inhibits subsequent proliferation and Ig secretion. TGF-beta may function as an autocrine growth inhibitor that limits B lymphocyte proliferation and ultimate differentiation.     

Regulation of colony-stimulating factor 1-dependent macrophage precursor proliferation by type beta transforming growth factor

Transforming growth factor (TGF) type beta, a potent growth modulator, has recently been shown to inhibit the proliferation and function of several types of immune cells. This report investigates the effect of human platelet purified TGF-beta on CSF-1-induced proliferation in liquid cultures. We used two cell types to study TGF-beta effects, bone marrow precursors and a c-myc partially transformed CSF-1-dependent macrophage cell line designated BMM-8. We found that CSF-1-dependent proliferation of both cell types was strongly inhibited by TGF-beta in a dose-dependent manner. Approximately 1.6 and 8 pM TGF-beta inhibited 50% of CSF-1 proliferation of the bone marrow precursors and BMM-8, respectively. Inhibition appeared to be reversible, as bone marrow and BMM-8 cells proliferated in response to CSF-1 after preincubation of the cells in TGF-beta. Interestingly, inhibition of hematopoietic cells was observed only after a lag period of 24 to 48 h after onset of cultures. TGF-beta inhibition was partially diminished when increasing amounts of CSF-1 were added to the cultures. TGF-beta inhibition did not involve secondary inhibitory factors such as IFN or PG, both of which have been previously shown to suppress CSF responsiveness. Finally, flow cytometric analysis of the cell cycle indicated that within 48 h, TGF-beta-treated BMM-8 cells were prevented from entering S phase. These results suggest that TGF-beta may play an important role in the negative regulation of macrophage production.     

Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-beta- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures

Both transforming growth factor (TGF-beta) and growth and development factor (GDF)-8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high-affinity insulin-like growth factor binding proteins (IGFBP 1-6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF-beta(1) or GDF-8 suppressed proliferation and increased production of IGFBP-3 protein and mRNA (P < 0.005). An anti-IGFBP-3 antibody that neutralizes the biological activity of IGFBP-3 reduced the ability of either TGF-beta(1) or GDF-8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF-beta(1) and GDF-8. These data show that IGFBP-3 plays a role in mediating the activity of either TGF-beta(1) or GDF-8 alone but not when both TGF-beta(1) and GDF-8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP-3 did not result in increased levels of phosphosmad-2. Since TGF-beta and GDF-8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP-3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP-3 also may be involved in regulating these processes in myogenic cells.     

Hepatocyte growth factor regulation of uterine epithelial cell transepithelial resistance and tumor necrosis factor alpha release in culture

Underlying stromal cells are essential for the normal development of epithelial cells (ECs) at mucosal surfaces. Recent studies from our laboratory have shown that uterine stromal cells regulate EC integrity, measured as transepithelial resistance (TER) as well as tumor necrosis factor (TNF) alpha alpha secretion by ECs in culture. Using stromal cells in coculture with polarized ECs grown on inserts, we found that stromal cells produce soluble mediators that increase TER and decrease TNFalpha secretion. The purpose of the present study was to identify the mechanisms whereby stromal cells exert their effects on uterine epithelium. We report that hepatocyte growth factor (HGF), a known mesenchymal growth factor that mediates EC proliferation, increases TER but, at the same time, decreases apical TNFalpha release. When ECs and/or stromal cells were incubated with anti-HGF or anti-HGF receptor (HGFR) antibody before HGF, the effects of HGF were blocked. These findings indicate that ECs express the HGFR at their basolateral surfaces and that HGFR mediates the effects of HGF on TER and TNFalpha. Neutralization of stromal cell secretions with antibodies for HGF and HGFR demonstrate that stromal-derived HGF is the mediator of EC TER. In contrast, neither anti-HGF antibody nor HGFR antibody had any effect on stromal cell-induced decreases in TNFalpha secretion. From these results, we conclude that stromal cell regulation of EC TER is mediated through the secretion of stromal HGF. Furthermore, because neutralization of stromal media failed to affect TNFalpha secretion, these findings suggest that other growth factors, in addition to HGF, affect EC cytokine production.     

F9 embryonal carcinoma cells can differentiate into endoderm-like cells

The mouse teratocarcinoma cell line, F9, has been used in many laboratories as the epitome of the “nullipotent” embryonal carcinoma cell line. However, careful inspection of F9 cultures reveals the presence of small numbers of cells which possess several properties of endoderm, particularly parietal endoderm, and which can be shown to derive from the embryonal carcinoma component. Furthermore, tumors of F9 cells include isolated patches of endoderm-like cells surrounded by a thick secretion resembling Reichert's membrane. The proportion of endoderm-like cells in F9 cultures can be increased to varying degrees by causing the cells to form aggregates and/or maintaining them at high density for several days, although the endoderm-like cells produced in these ways contribute very little to the formation of subcutaneous tumors from the resultant mixed cultures. Differentiated cell types other than endoderm are rarely observed in F9 monolayer or aggregate cultures, even after several weeks. Cloning studies support the view that most, if not all, F9 cells can differentiate, albeit at very low incidence.     

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.     

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.     

Differential responsiveness of myc- and ras-transfected cells to growth factors: selective stimulation of myc-transfected cells by epidermal growth factor.

To identify functional relationships between oncogenes and growth factors, we compared the effects of transfected myc and ras oncogenes on the responsiveness of Fischer rat 3T3 cells to three growth factors: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-beta). Control cells did not grow in soft agar under any conditions. ras-Transfected cells grew in soft agar under all conditions tested and were insensitive to the stimulatory effects of exogenous growth factors. These cells secreted elevated levels of both EGF-like factors and TGF-beta, suggesting that the lack of responsiveness of these cells to exogenous growth factors arose from autocrine stimulation. myc-Transfected cells displayed conditional anchorage-independent growth: they formed numerous colonies in soft agar in the presence of EGF but relatively few colonies in the presence of PDGF or TGF-beta. Secretion of EGF-like factors and TGF-beta by these cells was not elevated above that of control cells. These results suggest a model for the mechanism of cooperation between myc and ras oncogenes in which ras-like genes induce growth factor production, while myc-like genes increase the responsiveness of cells to these factors.     

Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.     

Transforming growth factor-beta receptor profiles of human and murine embryonic palate mesenchymal cells.

Cell signalling in the developing mammalian palate appears to involve various growth factors and hormones. An important developmental role for the transforming growth factor-beta (TGF-beta) class of growth factors is suggested by the immunolocalization of TGF-beta 1 in the palate during its ontogeny. This study examined the effects of TGF-beta stimulation of, as well as TGF-beta receptor profiles in, murine embryonic palate mesenchymal (MEPM) and human embryonic palate mesenchymal (HEPM) cells. Results showed that TGF-beta 1 (1 ng/ml) stimulated proliferation of HEPM cells and inhibited proliferation of MEPM cells in a dose-dependent manner. The time course of 125I-TGF-beta 1 binding to specific receptors was determined by incubating cells in the presence of 170 pM 125I-TGF-beta 1 for up to 4 h. In both cell types, at 37 degrees C, the binding of 125I-TGF-beta decreased linearly over 4 h, while at 4 degrees C, binding increased with time of incubation. Incubation of both cell types at 4 degrees C for 4 h, with increasing concentrations of 125I-TGF-beta 1, resulted in binding which demonstrated saturation kinetics. Scatchard analyses revealed one class of receptors for HEPM (K 32.3 pM) and MEPM (K 26.3 pM). However, SDS-PAGE analyses of 125I-TGF-beta chemically crosslinked to specific receptor sites revealed that both cell types contained the types I (65,000 Mr) and III (230,000 Mr) TGF-beta receptors while MEPM also contained the type II (86,000 Mr) receptor. Binding studies further demonstrated the ability of platelet-derived growth factor to transmodulate TGF-beta binding. These results indicate that the HEPM cell line and primary cultures of MEPM cells, although obtained from palates at similar developmental stages, are dramatically different in their responsiveness to TGF-beta and have disparate TGF-beta receptor profiles.