Extracellular calcium mimics the actions of platelet-derived growth factor on mouse fibroblasts.

Microprecipitates of calcium phosphate (CaPO4) can substitute for platelet-derived growth factor (PDGF) to stimulate the growth of cultured 3T3 cells. In two-part complementation assays, CaPO4 behaves as a PDGF-like "competence factor"--that is, the mitogenic response to CaPO4 is enhanced synergistically by "progression factors" contained in platelet-poor plasma. In studies described here, we show that early cytoplasmic and intranuclear events in the mitogenic response to CaPO4 are equivalent to those induced by PDGF. However, no net increase in tyrosine kinase activity of either the PDGF-alpha or PDGF-beta receptor is seen following exposure to CaPO4. Our data suggest that calcium acts within the cell, regulating events which normally proceed from activation of PDGF receptors. Alternatively, microprecipitates of CaPO4 could act externally by activating a growth factor receptor which escapes detection with available reagents.     

The effects of platelet-derived transforming growth factor beta on normal human diploid gingival fibroblasts

Studies of the effects of transforming growth factor (TGF) beta on normal human diploid gingival fibroblasts (HGF) have been carried out to determine possible physiological effects of this growth factor. Responses distinctly different from those characterized using established cell lines were observed. Whether alone, or in combination with EGF (2.5 ng/ml), human platelet-derived TGF-beta (0.1 ng/ml or 1.0 ng/ml) did not induce anchorage-independent growth of HGFs in soft agar assays. However, TGF-beta with EGF acted synergistically in promoting a 1.8-fold increase in anchorage-dependent proliferation of quiescent HGFs. At the same concentrations TGF-beta alone stimulated the incorporation of [35S]methionine into both cellular (cell-layer) and matrix (medium) proteins by as much as 3-fold and 1.7-fold respectively. Densitometric analysis of fluorographs of radiolabeled media proteins separated by SDS-PAGE revealed that the TGF-beta-stimulated protein synthesis was selective. However, synthesis of collagen, the major protein synthesized and secreted by HGFs, was stimulated by TGF-beta to the same extent as the average secreted protein. Protein synthesis and cell proliferation were significantly greater in subconfluent cells compared to confluent and multilayered cells. These effects are likely to reflect physiological activity of platelet-derived TGF-beta which may act to promote the wound healing response.     

Induction of NF-kappa B-like activity by platelet-derived growth factor in mouse fibroblasts.

Nuclear factor kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse fibroblasts by platelet-derived growth factor (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not 15 min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related factor because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like factor may participate in the expression of PDGF-inducible genes.     

Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts

The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.     

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts

Heparan sulfate is a molecule that possesses a large structural variability and which has been shown to inhibit the proliferation of fibroblasts in vitro. The aim of this study was to determine whether the anti-proliferative effects of heparan sulfate were exerted by regulation of the activity of the platelet-derived growth factor and/or of the platelet-derived growth factor receptors. Both l-iduronate-rich, anti-proliferative and the l-iduronate-poor, non-anti-proliferative heparan sulfate species, were incubated with confluent human embryonic lung fibroblasts for 24 h. The mRNA levels for PDGF-AA, PDGF-BB, and their receptors were measured. Binding studies were performed with [125I]-PDGF-BB and [125I]-EGF for 2 h at 4 degreesC in cultures preincubated with both types of heparan sulfate for 24 h. In separate experiments, cultures were incubated together with heparan sulfate and [125I]-PDGF-BB for 2 h at 4 degreesC. Increases of two- to threefold in the mRNA levels for both the alpha- and the beta-receptors of PDGF was obtained after treatment with both types of heparan sulfate, whereas the mRNA levels of both the PDGF-AA and the PDGF-BB were essentially unaffected. A sixfold increase in binding was only noted for [125I]- PDGF-BB in cultures pre-treated with the anti-proliferative heparan sulfate for 24 h, whereas no effect was noted with use of the non-anti- proliferative heparan sulfate. Incubating the [125I]-PDGF-BB and the anti-proliferative heparan sulfate together for 2 h resulted in a smaller, threefold increase in binding. This indicates that the anti- proliferative heparan sulfate both stabilizes and increases expression of the PDGF receptors. To investigate whether the increased number of PDGF receptors could affect cell activity, cells were preincubated with anti-proliferative heparan sulfate and then treated with PDGF-BB. This resulted in an increase in mitogenicity compared to cells treated only with PDGF-BB. Neither an increase in binding for [125I-EGF] nor an increase in the mitogenic response of EGF could be observed in cultures pre-treated with the anti-proliferative heparan sulfate. The results indicate that the extracellular matrix itself may regulate important biological phenomena such as cell proliferation and matrix production through affecting the expression of receptors of PDGF, which initiate both stimulatory and inhibitory signals.      

Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor

One of the earliest effects of platelet-derived growth factor (PDGF) on human fibroblasts in culture is an induction of membrane ruffling. The morphology of the ruffles induced by PDGF is unique in that they form circular arrangements on the dorsal side of the cells. Here we report that the induction of circular ruffle arrangements is an effect specific for PDGF, dose-dependent and inhibitable by anti-PDGF antibodies. We have attempted to utilize this effect to design a rapid and sensitive bioassay for PDGF. The "membrane ruffling assay" is compared with other methods to measure PDGF and its specificity with regard to the different dimeric forms of PDGF is discussed. Introduction of Ca2+ into the cells via the Ca2+ ionophore A23187 or the addition of the tumor-promor 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a stimulator of protein kinase C, does not induce circular ruffle formations on human fibroblasts, neither does the addition of the combination of these two agents. However, addition of TPA almost completely inhibits PDGF-induced circular ruffle formations. Further, we find a shift in the time-course of the PDGF-induced circular ruffle formations by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases. This may indicate the involvement of protein phosphorylation in the regulation of PDGF-induced membrane ruffling.     

Characterisation and application of antibodies specific for the long platelet-derived growth factor A and B chains

The platelet-derived growth factor (PDGF) family comprises important mitogens for mesenchymal cells. The active dimeric form of PDGF consists of four structurally related A, B, C, and D chains. All PDGF-variants bind to PDGF-receptors. The A and B chains occur with and without basic C-terminal amino acid extensions as long (A(L) and B(L)) and short (A(S) and B(S)) isoforms. PDGF-A and -B form homo- or heterodimers. The biological relevance of short and long isoforms is unknown, although it may relate to different affinities for glycosaminoglycans of the cell glycocalix and intercellular matrix. Commercially available anti-PDGF-A and anti-PDGF-B antibodies cannot discriminate between the short and the long isoforms. Thus, to investigate the function of the long and short isoforms, we raised antibodies specific for the long A and B chain isoforms. The antibodies were affinity-purified and their properties analysed by surface plasmon resonance. Inhibition studies with different PDGF homodimers and dot-blot studies proved their high specificity for the respective isoforms. Both antibodies recognised the target PDGF homodimers complexed to the glycocalix of human arterial smooth muscle cells and human monocyte-derived macrophages. By using these specific antibodies, we were able to confirm at the protein level the synthesis of PDGF-A and -B during differentiation of human monocyte-derived macrophages and to demonstrate the presence of the PDGF-A(L) and PDGF-B(L) isoforms in human arterial tissue.     

Effects of platelet-derived growth factor on phosphorylation of the epidermal growth factor receptor in human skin fibroblasts

Heterologous regulation of the epidermal growth factor (EGF) receptor by platelet-derived growth factor (PDGF) was studied in FS4 human skin fibroblasts. The addition of PDGF to FS4 cells inhibited high affinity binding of 125I-EGF and stimulated phosphorylation of the EGF receptor. Phosphopeptide analysis by high performance liquid chromatography revealed that PDGF treatment of cells increased phosphorylation at several distinct sites of the EGF receptor. However, PDGF did not stimulate phosphorylation of threonine 654, a residue previously shown to be phosphorylated when protein kinase C is activated. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated phosphorylation of the same peptides from the EGF receptor as PDGF, and, in addition, induced phosphorylation of threonine 654. TPA inhibited both high and low affinity 125I-EGF binding by these cells. PDGF treatment of cells had no effect on EGF-dependent, tyrosine-specific autophosphorylation of the receptor, whereas TPA treatment was inhibitory. TPA, but not PDGF, stimulated phosphorylation of a Mr = 80,000 protein, known to be a substrate for protein kinase C, even though PDGF appeared to mediate breakdown of phosphoinositides. These data suggest that regulation of EGF receptor function by PDGF and TPA are distinct in these cells, even though some elements of regulation are shared. The results differ from those previously reported for a human lung fibroblast isolate, indicating that cell type-specific differences may exist in metabolism of the EGF receptor.     

Modulation of muscarinic-receptor expression in human embryonic lung fibroblasts by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is known to have regulatory control of a large number of cellular components, including various receptors. We show that muscarinic acetylcholine receptors of the m2 subtype on CCL 137 human fibroblasts in culture are affected by PDGF treatment. A time-dependent down-regulation is observed in steady-state RNA levels, followed by a decrease in ligand-binding capacity. Minimum RNA levels are attained at 11 h; minimum binding capacity is observed after 24 h of treatment. To our knowledge, this is the first example of negative gene control by PDGF.     

Effect of the growth conditions on the expression of cell-surface-associated platelet-derived growth factor receptors in mouse fibroblasts

The conditions affecting the appearance and disappearance of platelet-derived growth factor (PDGF) receptors from the pool of active cell surface-associated receptors were studied. Receptor molecules were revealed in intact Swiss 3T3 fibroblasts by their ability to bind 125I-labeled PDGF and, due to their property to become phosphorylated in tyrosine following ligand binding, by antibodies to phosphotyrosine. PDGF receptor molecules were found to be quite scarce in exponentially growing fibroblasts as compared to quiescent cells. When growing cells were either shifted to a medium containing plasma or received suramin in the culture medium, cell surface-associated PDGF receptors largely increased. This process required about 12 h. Incubation of quiescent cells in serum, but not in plasma, induced a slow decrement of ligand-activatable receptors. In the presence of PDGF the rate of receptor removal from the cell surface was very rapid and was a function of the PDGF concentration. Quiescent cells deprived of cell-surface receptors by incubation with PDGF reexpressed PDGF receptors in about 14 h.     

Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts

The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were super-induced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c-myc mRNA level, indicating that the effect of PDGF on c-myc expression is not caused by a "hit and run," mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c-fos and c-myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.     

Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor

Previous studies of Rac1 in fibroblasts have used dominant negative constructs, which may have nonspecific effects. We used a conditional Rac1 allele to critically examine Rac1 function in mouse fibroblasts. Lack of Rac1 had dramatic effects on nonconfluent cells, which were elongated and had extensive blebbing, but no lamellipodia or ruffle formation. However, Rac1-null fibroblasts translocated using pseudopodia-like protrusions without lamellipodia, migrating toward a platelet-derived growth factor (PDGF) gradient as efficiently as their wild-type counterparts. Rac1-null fibroblasts closed wounds in vitro and spread on a fibronectin substrate, although at a slower rate than wild-type cells. However, Rac1-null cells were markedly impaired in proliferation, with a defect in G1 to S transition, although they were capable of surviving in culture for more than 2 wk. These results refine our understanding of the functions of Rac1, indicate that lamellipodia formation is not required for cell motility, and show that PDGF-induced chemotaxis can occur in the absence of both lamellipodia and Rac1.     

Mitogenic action of tumor necrosis factor in human fibroblasts: interaction with epidermal growth factor and platelet-derived growth factor

We have previously shown that tumor necrosis factor (TNF) can increase the number of epidermal growth factor (EGF) receptors on human FS-4 fibroblasts and that this increase may be related to the mitogenic action of TNF in these cells. Here we show that TNF stimulated the growth of FS-4 fibroblasts in a chemically defined, serum-free medium in the absence of EGF. Anti-EGF receptor antibody, which blocked the mitogenic effects of EGF in FS-4 cells, did not inhibit the mitogenic action of TNF in serum-free or serum-containing medium, indicating that EGF or an EGF-like molecule was not responsible for the mitogenic effects of TNF. However, the simultaneous addition of TNF and EGF to cells grown in serum-free medium resulted in a synergistic stimulation of DNA synthesis and cell growth. The actions of TNF and EGF were also examined in growth-arrested FS-4 cells and were compared with the action of platelet-derived growth factor (PDGF). In the absence of other growth factors, TNF was a relatively weak mitogen in growth-arrested cells, compared with EGF or PDGF. Nevertheless, TNF synergized with EGF or high doses of PDGF in stimulating DNA synthesis. Furthermore, antibodies specific for TNF or the EGF receptor were used to selectively inhibit the actions of these two factors, after specific incubation periods, in growth-arrested cells treated concurrently with EGF and TNF. To produce an optimal stimulation of DNA synthesis, EGF had to be present for a longer period of time than TNF. We conclude that in their synergistic action on growth-arrested FS-4 cells, EGF was responsible for driving the majority of the cells into S phase, while TNF appeared to make the cells more responsive to the mitogenic action of EGF. The findings indicate that TNF can cooperate with, and enhance the actions of, EGF in promoting DNA synthesis and cell division.     

Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF β-receptors were found to be enhanced in old donor cells strains, whereas the PDGF α-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts. © 1994 Wiley-Liss, Inc.     

Different effects of platelet-derived growth factor isoforms on rat vascular smooth muscle cells

The response of rat aortic smooth muscle cells to all three isoforms of platelet-derived growth factor (PDGF) was studied. 5,000 binding sites/cell were estimated for rPDGF-AA (Kd 0.22 nM), 45,000 for rPDGF-AB and (Kd 0.4 nM), and 31,000 for rPDGF-BB (Kd 0.29 nM). rPDGF-AB and -BB stimulated effectively [3H]thymidine incorporation, inositol 1,4,5-trisphosphate release, diacylglycerol productions, [Ca2+]i increase, and pHi changes at concentration in the range from 3 to 10 ng/ml. The extent of DNA synthesis stimulated by rPDGF-AA varied considerably, and in all cases higher concentrations than 10 ng/ml were required. rPDGF-AA did not stimulate inositol-1,4,5-trisphosphate release, [Ca2+]i increase or pHi changes but induced the production of diacylglycerol, although with a different kinetic compared with that observed with rPDGF-AB or -BB. Apparently rPDGF-AA acts via a different mechanism, generating diacylglycerol without the release of inositol-1,4,5-trisphosphate.     

Mitogenic effect of transforming growth factor beta 1 on human fibroblasts involves the induction of platelet-derived growth factor alpha receptors

Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF-beta on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF-beta 1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF-beta 1 potentiates DNA synthesis in PDGF-stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF-beta 1 enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF-AA and 125I PDGF-BB, consistent with the increased expression of the alpha type PDGF receptor. The induction of PDGF alpha receptor subunits by TGF-beta may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.     

Cell cycle regulation of human diploid fibroblasts: possible mechanisms of platelet-derived growth factor

Cell-cycle regulation of human diploid fibroblasts (HDF) is located in the proximal half of G1, designated G1-pm (G1-postmitosis). In order to traverse this subphase, cells require serum factors or PDGF. However, when cells have traversed into the distal half of G1, designated G1-ps (G1-pre-DNA synthesis), they become independent of serum or PDGF and progress through the remainder of the cell cycle at an invariable rate. From this, it follows that a specific G1-pm block can be induced by serum depletion. A similar G1-pm block could also be induced by a moderate inhibition of overall protein synthesis following treatment with CHM. Even this block could be prevented by the addition of PDGF, suggesting that a high level of protein synthesis in itself is not necessary for sustaining cell-cycle traverse. Nevertheless, a critical accumulation of some specific proteins might be required for the G1-pm/G1-ps-transition. However, the underlying mechanisms of modulation of the accumulation of such proteins by PDGF must involve alternative regulatory events (e.g., gene expression, protein stabilization) rather than protein synthesis. Among the possible cell cycle-regulatory proteins, the present study focused on 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. This enzyme is regulated by various kinds of control mechanisms and regulates the biosynthesis of sterols and nonsterol isoprenes, some of which are proposed to be necessary for mammalian cell growth (Brown and Goldstein, 1980). The present results suggest that regulation of HMG CoA reductase may be involved in the control of the G1-pm/G1-ps-progression in HDF.     

Crystal structure of human platelet-derived growth factor BB.

The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution. The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites.     

Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.     

Effects of platelet-derived growth factor on human and mouse osteoblastic cells isolated from the trabecular bone surface.

We show here that purified platelet derived growth factor (PDGF) stimulates DNA synthesis in normal endosteal mouse and human osteoblastic cells isolated by selective migration from the trabecular bone surface. Maximum DNA synthesis as measured by (3H)-thymidine incorporation into DNA was increased at 50 ng/ml PDGF (48-72 hours). In both species, the effect of PDGF (25 ng/ml) was lower than the mitogenic effect of 10% FCS. We found that the mitogenic effect of PDGF on human trabecular cells decreased with the number of cell passages. DNA synthesis was increased about 4-fold by PDGF (25 ng/ml) in early passaged cells that expressed low basal growth rate and high osteocalcin production in basal conditions and in response to 1,25(OH)2 vitamin D, whereas DNA synthesis was increased 1.2 fold by PDGF in late passaged cells that showed high basal growth rate and low osteocalcin release in absence or presence of 1,25(OH)2D. PDGF alone had no effect on osteocalcin production. These results indicate that PDGF has mitogenic effect on normal mouse and human osteoblastic cells lining the trabecular bone surface and that the responsiveness to PDGF of human trabecular cells varies with the stage of differentiation.