Hepatocyte growth factor releases epithelial and endothelial cells from growth arrest induced by transforming growth factor-beta1

Human lung fibroblasts and Mv1Lu mink lung epithelial cells were used as a model to study the role of extracellular matrix in epithelial-mesenchymal interactions. Extracellular matrices of fibroblasts were found to contain growth promoting activity that reduced the sensitivity of Mv1Lu cells to the growth inhibitory effects of transforming growth factor-beta (TGF-beta). The majority of the activity was identified as hepatocyte growth factor/scatter factor (HGF) by inhibition with specific antibodies and by reconstitution of the effect by recombinant HGF. HGF induced cell proliferation when contact-inhibited Mv1Lu cells were trypsinized and plated in the presence of TGF-beta1. The effect was valid also in assays where Madin-Darby canine kidney epithelial cells or bovine capillary endothelial cells were used. The multiplication of chronically TGF-beta1 inhibited Mv1Lu cells was also induced by HGF. In addition, HGF induced anchorage independent growth of Mv1Lu cells that was refractory to TGF-beta1 growth inhibition. Immunoprecipitation analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2, but not the induction of p21, by TGF-beta1. Since both TGF-beta1 and HGF require proteolysis for activation, the results imply that proteolytic activity of epithelial and endothelial cells directs their responses to signals from mesenchymal-type extracellular matrices, and that during development, matrix-bound growth and invasion promoting and suppressing factors are activated in a coordinated manner.     

Activation of the insulin-like growth factor-I receptor inhibits tumor necrosis factor-induced cell death

The effect of insulin-like growth factor (IGF) on tumor necrosis factor (TNF)-induced cell killing was determined for mouse BALB/c3T3 fibroblasts in vitro. Cells maintained in 0.5% fetal bovine serum (FBS) were killed by TNF within 6 h in a concentration-dependent manner, an effect that was prevented by IGF-I. TNF-induced cytotoxicity of 3T3 cells that overexpress the human IGF-I receptor (p6 cells) was prevented by IGF-I alone in the absence of serum. TNF-induced cell death was associated with the morphologic features of apoptosis and the release of low-molecular-weight DNA, both of which were prevented by IGF-I. Neither epidermal growth factor (EGF) nor platelet-derived growth factor (PDGF) protected p6 cells from TNF-induced apoptosis. The specific protective action of the IGF-I receptor was demonstrated further by the marked sensitivity to TNF of embryo fibroblasts derived from mice with targeted disruption of the IGF-I receptor (R-cells) but not of fibroblasts derived from wild-type littermates or R-cells transfected with the cDNA for the human IGF-I receptor. Cycloheximide or actinomycin D markedly reduced the protection offered by IGF-I. IGF-I protection of BALB/c3T3 cells persisted for up to 5 days in the presence of PDGF and EGF, whereas IGF-I lost its effectiveness after 2 days in the absence of growth factors. IGF-I did not prevent TNF-induced release of arachidonic acid. The results demonstrate a specific role for the IGF-I receptor in the protection against TNF cytotoxicity. This action of the IGF-I receptor is mediated by protective cytosolic proteins that exhibit a high rate of turnover and whose levels are regulated principally by factors within serum other than IGF-I.     

Factors required for bone marrow stromal fibroblast colony formation in vitro

Marrow stromal fibroblasts (MSFs) are essential for the formation of the haemopoietic microenvironment and bone; however, regulation of MSF proliferation is poorly understood. MSF colony formation was studied in primary mouse and human marrow cell cultures. After a brief exposure to serum, MSF colony formation occurred in the absence of both serum and non-adherent marrow cells, if medium conditioned by marrow cells was present (serum-free conditioned medium, SF-CM). In mouse and human cultures stimulated to proliferate by SF-CM, neutralizing antibodies against PDGF, TGF-beta, bFGF and EGF specifically suppressed MSF colony formation. The degree of supression was species-dependent, with the most profound inhibition achieved in mouse cultures by anti-PDGF, anti-bFGF and anti-EGF, and in human cultures by anti-PDGF and anti-TGF-beta. Serum-free medium not conditioned by marrow cells (SFM) did not support MSF colony formation. In mouse cultures in SFM, human recombinant bFGF and bovine natural bFGF were able to partially substitute for the stimulating effect of SF-CM. Other growth factors, including TGF-beta1, TGF-beta2, PDGF, EGF, IL-6, IGF-I and IGF-II, showed no activity when tested alone. In human cultures in SFM, none of the growth factors, alone or in combination, stimulated MSF colony formation. Mouse and human MSFs grown in SF-CM formed bone and a haemopoietic microenvironment when transplantated into immunodeficient mice in vivo, and therefore were functionally equivalent to MSFs generated in the presence of serum. These data indicate that stimulation of the initial proliferation of an MSF precursor cell is complex, and requires participation of at least four growth factors: PDGF, bFGF, TGF-beta and EGF. In addition, mouse and human MSF precursor cells have different requirements for each of the growth factors.     

Platelet-derived growth factor stimulates the release of protein kinase A from the cell membrane

The mitogenic action of growth factors involves the stimulation of intracellular protein kinases. In this report we have characterized the major protein kinase released from Balb/c 3T3 and normal rat kidney plasma membranes by the action of platelet-derived growth factor (PDGF). PDGF appears to stimulate the release of approximately 10 proteins, at least one of which is a kinase capable of phosphorylating proteins on Ser or Thr (as determined by the lability of the phosphate to alkali treatment). More than 90% of the Ser/Thr kinase activity was inhibited by PKI5-22, a specific peptide inhibitor of the cAMP-dependent protein kinase (PKA). We used immunoblotting to confirm that the kinase released in response to PDGF was PKA. cAMP also stimulated the release of PKA, and the set of protein substrates phosphorylated was similar following PDGF or cAMP stimulation. Interestingly, in the presence of a cAMP analogue ((Rp)-cAMPS), cAMP could not induce dissociation of PKA from the membranes, whereas stimulation by PDGF increased the level of PKA activation. Furthermore, unlike Swiss 3T3 cells, neither Balb/c 3T3 fibroblasts nor normal rat kidney cells accumulate cAMP in response to PDGF, yet the level of PKA in the cytosol of these intact cells increases in response to PDGF. Thus, it appears as though PDGF activation of the membrane-associated form of the PKA holoenzyme occurs by a mechanism independent of an elevation in cAMP levels.     

Characterization of insulin-like growth factor binding proteins and regulation of IGFBP3 in human mesangial cells

IGF-I regulates renal growth and development. Insulin-like growth factor binding proteins (IGFBPs) are synthesized by the kidney and may modulate the local autocrine and/or paracrine actions of IGF-I. We have previously demonstrated that mesangial cells (MC) release IGF-I and IGF-binding activity; however, the specific IGFBPs produced by these cells and the factors involved in their regulation are unknown. We examined MC for expression of IGFBP-1 to -6 mRNAs and proteins. RNase protection assays using total RNA demonstrated that MC express all of the IGFBPs. [125I]IGF-I Western ligand blot of conditioned medium demonstrated that MC release IGFBPs of 24, 29, 32 kDa, and a doublet at 46 kDa, consistent with IGFBP-4, -5, -2 and -3, respectively. IGFBP species of 28 and 34 kDa were also detected. Since IGF-I and TGF-beta are implicated in glomerular hypertrophy and matrix expansion, we tested their effect on IGFBPs released by MC. IGF-I (100 ng/ml), TGF-beta (2 ng/ml) and forskolin (10(-5) M) differentially regulated the abundance of IGFBPs released in the conditioned medium in a time-dependent manner. IGF-I and TGF-beta were potent inducers of the release of IGFBP3 protein; however, TGF-beta, but not IGF-I, increased IGFBP3 mRNA levels. Recombinant IGFBP3 was tested for its effect on IGF-I-induced mitogenesis. IGFBP3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner with a peak effect observed at 50 nM IGFBP3. Although TGF-beta is a potent inhibitor of IGF-I-stimulated DNA synthesis, this effect is not mediated via IGFBPs. Expression of IGFBP-1 to -6 by MC suggests that these proteins may modulate IGF-I bioavailability in the glomerulus. IGF-I itself, TGF-beta and cAMP agonists may indirectly modulate the effects of IGF-I via the release of IGFBPs by MC.     

Activation of a calcium-permeable cation channel CD20 expressed in Balb/c 3T3 cells by insulin-like growth factor-I

CD20 functions as a calcium-permeable cation channel. When expressed in Balb/c 3T3 cells, CD20 accelerates the G1 progression induced by insulin-like growth factor-I (IGF-I). To further characterize how CD20 modulates the action of IGF-I, we investigated whether the activity of CD20 channel was affected by IGF-I. In quiescent cells expressing CD20, IGF-I increased cytoplasmic free calcium concentration, [Ca2+]c, which was reversed by the removal of extracellular calcium. In contrast, IGF-I did not increase [Ca2+]c in cells that did not express CD20. In perforated patch clamp recordings, addition of IGF-I to the bath solution augmented the Ca2+ permeability, which was reversed by anti-CD20 antibody. In cell-attached patch, calcium-permeable channel activity with unitary conductance of 7 picosiemens was detected, which was abolished by anti-CD20 antibody. The single channel activities were markedly enhanced when IGF-I was included in the pipette solution, whereas IGF-I added to the bath solution was ineffective. When cells were first exposed to pertussis toxin, activation of the channel by IGF-I was blocked. Transfection of cDNA for Gip2, a constitutive active form of alphai2, activated the CD20 channel. These results indicate that the CD20 channel is regulated by the IGF-I receptor by a mechanism involving pertussis toxin-sensitive G protein.     

Insulin-like growth factor II stimulates cell proliferation through the insulin receptor

R- cells are 3T3-like fibroblasts generated from mouse embryos nullizygous for a targeted disruption of the genes encoding the type 1 insulin-like growth factor (IGF) receptor (IGF1R). These cells fail to proliferate in serum-free medium supplemented with purified growth factors, in contrast to their wild-type counterparts. However, when R- cells overexpress the insulin receptor from a stably integrated plasmid, R-/IR cells, they become capable of growing in serum-free medium supplemented solely with insulin or IGF-II, but not with IGF-I. Moreover, the introduction into R-/IR cells of an additional plasmid expressing IGF-II causes these cells to proliferate in serum-free medium without growth factor supplementation. From these results, we conclude that IGF-II can stimulate cell proliferation not only through its cognate IGF1R but also through the insulin receptor.     

Peptide growth factors in the prostate as mediators of stromal epithelial interaction

Peptide growth factors play a role in the maintenance of normal prostatic growth and differentiation (Fig. 2). It seems likely that the androgen sensitivity of human prostate is mediated by the production of peptide growth factors from stromal cells which act as the direct intermediate of androgen action on epithelial cells. TGF-beta 1 inhibition of epithelial cells is opposed by the stimulatory action of EGF, IGF and FGFs to maintain an equilibrium of epithelial cell numbers. The indirect mitogenic action of androgens appear to act by down-regulation of TGF-beta 1 and possibly EGF receptors. There is also interaction with the effects of IGF-II, produced by prostatic stromal cells and acting on epithelial cells to increase proliferation. The growth of normal prostatic fibroblasts is under the control of bFGF and TGF-beta 1. However, although our understanding of the actions of these growth factors in the normal prostate has improved over the last decade, their role in the development and maintenance of prostate cancer is less clearly defined. TGF-beta 1, classically considered to be inhibitory for epithelial cells, may be up-regulated in prostatic tumours, stimulating growth. Alternatively, autocrine production of such growth factors by tumour cells may lead to loss of inhibitory effects from exogenous TGF-beta 1, a mechanism also witnessed with TGF-alpha and bFGF. The role of EGF in the development of prostate cancer is confusing because results from the use of different cell types and experimental conditions is contradictory. It may be that a switch in the production of the predominant EGFr ligand from EGF to TGF-alpha is an important feature in the development and maintenance of the malignant phenotype. The presence of TGF-alpha autocrine loops has been shown clearly in some tumour cell lines. This switch in the production of a particular ligand may also be a feature of IGFs in prostate cancer. IGF-II may be replaced by IGF-I during malignant progression, both of which are able to act via the type 1 receptor. This change in IGF expression appears to be accompanied by altered expression of the IGF-BP2, with less detectable within prostatic tissues but elevated serum levels [58]. Basic FGF is normally produced by prostatic fibroblasts but is also produced by some prostatic cancer cell lines [64]. However, as with all growth factors, the expression of the bFGF protein and its receptor is dependent on the cell line examined. The autocrine and paracrine control of normal and abnormal prostatic growth by growth factors is important in determining their role in the development and maintenance of prostate cancer. Better understanding of such mechanisms is essential for the development of novel therapeutic strategies in the control and treatment of prostate cancer.     

Insulin-like growth factor (IGF)-I and -II and IGFBP secretion by ovine satellite cell strains grown alone or in coculture with 3T3-L1 preadipocytes

The current study was designed to examine the effects of muscle and fat stem cell coculture on the secretion of insulinlike growth factor (IGF)-I and -II and IGF binding proteins (IGFBP) by these cells. Two sheep satellite cell strains with negligible or high potential for differentiation (10A and 0(1), respectively) were placed in coculture with 3T3-L1 preadipocytes using a filter support to separate the two cell types. Media conditioned by the cells grown alone or in coculture were analyzed for IGFs by RIA or IGFBPs by ligand blotting. The numbers of satellite cells and preadipocytes declined throughout the 5-d culture period, although coculture slowed the 3T3-L1 decline but hastened the satellite cell decline. The satellite cell strains and 3T3-L1 cells secreted small amounts of IGF-I (< or = 2 ng/ml) and IGF-II (< 10 ng/ml) over the 5-d culture period. Coculture did not increase the amount of IGF-I and -II in conditioned media. The lowly differentiating 10A cells secreted barely detectable amounts of the low molecular weight IGFBP-3 subunit (34 kDa), IGFBP-2 (28 kDa), and IGFBP-4 (18 kDa). Coculture of 10A and 3T3-L1 cells potentiated secretion of IGFBP-2 and -3. Strain 0(1), which readily differentiates, secreted high levels of both IGFBP-3 subunits (34 and 39 kDa) and IGFBP-2 (28 kDa), as well as significant amounts of the 18 kDa IGFBP-4. Coculture did not alter IGFBP secretion of 0(1) cells. This study showed that while IGF-I and -II levels in media conditioned by sheep satellite cell strains are low and relatively invariant, the intensity and complexity of IGFBP patterns increases with time in culture and with the potential for differentiation of the satellite cell strains. Coculture with preadipocytes appeared to potentiate IGFBP secretion while reducing satellite cell viability.     

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.     

CD44 protein levels and its biological activity are regulated in Balb/c 3T3 fibroblasts by serum factors and by transformation with the ras but not with the sis oncogene

CD44s (standard isoform) levels and hyaluronan-binding activity were investigated in Balb/c 3T3 cells and their derivatives transformed with ras or sis oncogenes as a function of serum concentration in the medium. 3T3 cells contained low levels of CD44 and did not bind hyaluronan when grown in medium containing 0.5 or 10% serum. In 5% serum, however, the cells had much higher levels of CD44 and were able to bind hyaluronan. CD44 levels also increased in 3T3 cells restimulated with either 5 or 10% serum after prior maintenance in low serum. In cells restimulated with 5% serum, high levels of CD44 were sustained for at least 72 hr. In cells restimulated with 10% serum, however, the increase in CD44 levels reverted by 48 hr. Transformation of 3T3 cells with ras (but not with sis) oncogene rendered CD44 levels insensitive to serum modulation: ras-transformed cells contained high levels of CD44 and bound hyaluronan at all serum concentrations and at all time points tested. Sis-transformed cells behaved like 3T3 cells in these modulatory changes. Platelet-derived growth factor (PDGF), when supplementing 0.5% serum, mimicked the effects of serum on the levels and hyaluronan-binding capacity of CD44 in 3T3 cells and the CD44-upregulating activity of serum was neutralized by incubation with anti-PDGF antibodies. These data demonstrate that serum factors, specifically PDGF, mediate regulation of CD44 levels in BAlb/c 3T3 cells and that transformation of 3T3 cells by ras renders CD44 expression insensitive to the modulating effects of serum in vitro. These results correlate with the metastatic capacity of these cells in vivo.     

Detergent solubility defines an alternative itinerary for a subpopulation of PDGF beta receptors

Current models of platelet-derived growth factor (PDGF) beta receptor itinerary are based upon the properties of receptors recovered from nonionic detergent-solubilized cellular extracts. Comparing several commonly used cell extraction procedures, we have determined that up to 50% of immunoreactive PDGF beta receptors, reside in a Triton X-100 insoluble pool in a wide distribution of cultured cell lines, including Balb/c-3T3, NIH 3T3, and Swiss fibroblasts, primary murine and human fibroblasts, and primary human glial cells. Many properties of Triton insoluble receptors are distinct from the well-characterized PDGF beta receptors, including 1) delayed arrival of newly synthesized receptors into the Triton insoluble fraction, 2) prolonged half-life in the presence of PDGF, 3) increased abundance with increasing cell density, 4) inaccessibility to modification by extracellular compartment enzymes, 5) cofractionation with cytoskeletal proteins, and 6) a higher basal tyrosine phosphorylation state. PDGF stimulates accumulation of tyrosine phosphorylated PDGF beta receptors in the Triton X-100 insoluble fraction. Cell surface PDGF beta receptors modified by enzymatic desialylation redistribute to the insoluble fraction. These findings distinguish the itinerary of a large subpopulation of PDGF beta receptors from those characterized previously. Receptors in this fraction represent a long-lived tyrosine phosphorylated population that may effect responses for extended periods following ligand activation.     

Growth regulatory effects of transforming growth factor-beta 1 and interleukin-2 on IL-2 dependent CD4+T lymphoblastoid cell line

Transforming growth factor-beta 1 (TGF-beta 1) is an immuno-modulatory cytokine which has been shown to modulate the activity of T and B cells. We show here that human TGF-beta 1 inhibited stationary cultures of IL-2 dependent CD4+ bovine lymphoblastoid T cells (BLTC) by down-regulating their IL-2 receptor (IL-2R) expression, arresting cells in the G0/G1 compartment of the cell cycle, and inducing these cells to undergo apoptosis. These events were reversed by the addition of a minimal concentration of IL-2 (2U/ml). In the presence of exogenous IL-2, TGF-beta 1 was found to augment the proliferative response of BLTC through up-regulation of IL-2R expression, allow progression of normal cell cycle, and significantly prevent apoptosis. Our data clearly show that IL-2 and TGF-beta 1, when present alone, have contrasting effects on BLTC. TGF-beta 1 down regulates events that are associated with IL-2 mediated signal. But when present together, IL-2 and TGF-beta 1 upregulate activation signals and proliferation of rapidly dividing CD4+T cells.     

Peptide-like substances in sheep amniotic fluid which regulate proliferation of BP-A31 cells

Cell proliferation and differentiation of developing fetus is influenced by hormones as well as insulin-like growth factors and their binding proteins contained in amniotic fluid. Our purpose was to study the actual mitogenic activity of proteins and peptides present in the sheep amniotic fluid. The cell cycle regulatory activity was estimated by using mouse fibroblasts BP-A31 as target cells. The whole amniotic fluid was inactive. However, after removal of small molecules on Sephadex G-10, the fraction eluted in the void volume (M(r) > or = 0.7 kDa) was able to induce the cell division cycle in a significant proportion of quiescent fibroblasts (Fig. 1, fraction A; Fig. 2). By further gel chromatography of this active fraction at acidic condition on Sephadex G-50, two components with mitogenic activity were separated. One component was eluted immediately after the void volume of the column, the other one was coeluted with 125I-IGF-I (Fig. 3). The functional characteristics of mitogenic signal of both components (sensitivity to mitogenic effectors) were similar to those of IGF-I and insulin (Fig. 4). We suppose that a component with higher molecular weight eluted in the vicinity of the void volume of Sephadex G-50 represents probably IGFBPs or other similar proteins.     

Deficient processing and activity of type I insulin-like growth factor receptor in the furin-deficient LoVo-C5 cells

To investigate endoproteolytic processing of the type I insulin-like growth factor receptor (IGF-IR), we have examined its structure and activity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation experiments using the monoclonal anti-IGF-IR antibody (alpha-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed alpha/beta pro-receptor. A small amount of successfully cleaved alpha/beta heterodimers was also produced, indicating a residual endoproteolytic cleavage activity in these cells. In vitro, a soluble form of recombinant furin was able to cleave the pro-IGF-IR (200 kDa) into alpha-subunit (130 kDa) and beta-subunit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding capacity, 5 x 10(3) sites/cell; Kd, 1.9 nM for IGF-I and 7.0 nM for IGF-II). These findings in LoVo-C5 contrast with those in HT29-D4 cells, which have active furin, and where IGF-IR (2.8 x 10(4) sites/cell) was fully processed. Moreover, the 200-kDa pro-IGF-IR of LoVo-C5 was unable to induce intracellular signaling, such as beta-subunit tyrosine autophosphorylation and insulin-related substrate-1 tyrosine phosphorylation. Flow immunocytometry analysis using alpha-IR3 antibody indicated that LoVo-C5 cells expressed 40% more receptors than HT29-D4 cells, suggesting that in LoVo-C5 cells only the small amount of mature type I IGF-IR binds IGFs with high affinity. To provide evidence for this idea, we showed that mild trypsin treatment of living LoVo-C5 cells partially restored alpha/beta cleavage of IGF-IR, and greatly enhanced (6-fold) the IGF-I binding capacity of LoVo-C5 cells, but did not restore IGF-IR signaling activity. Moreover, LoVo-C5 cells were totally unresponsive to IGF-I in terms of cell migration, in contrast to fully processed IGF-IR-HT29-D4 cells. Our data indicate that furin is involved in the endoproteolytic processing of the IGF-IR and suggest that this posttranslational event might be crucial for its ligand binding and signaling activities. However, our data do not exclude that other proprotein convertases could participate to IGF-IR maturation.     

Paracrine stimulation of human renal fibroblasts by proximal tubule cells

Paracrine stimulation of human renal fibroblasts by proximal tubule cells. BACKGROUND: Interstitial fibrosis strongly predicts the degree and progression of renal failure in human renal disorders. Since active fibrosis tends to initially occur in a peritubular distribution, the possibility that human proximal tubule cells (PTC) relay fibrogenic signals to neighboring cortical fibroblasts was examined in vitro. METHODS: Cell proliferation (cell counts and thymidine incorporation), total collagen synthesis (proline incorporation), matrix metalloproteinase (MMP) activity (gelatin zymography), and autocrine secretion of insulin-like growth factor-I (IGF-I) were measured in primary cultures of human cortical fibroblasts cocultured with PTC or exposed to PTC-conditioned media (PTCCM). RESULTS: Cell numbers and thymidine incorporation rates were increased in cortical fibroblasts cocultured with PTC (136.4+/-7.3% and 119.3+/-8.2% of control values, respectively, P < 0.05) or incubated in PTC-CM (114.0+/-5.9%, P < 0.05 and 146.7+/-13.3%, P < 0.05, respectively). PTC-CM stimulated cortical fibroblast collagen synthesis (13.5+/-1.0% vs. 10.8+/-0.7%, respectively, N = 24, P < 0.05) and MMP-2 and MMP-9 secretion. Cortical fibroblast secretion of IGF-I binding protein-3 (IGFBP-3), which in turn modulates the autocrine and paracrine actions of IGF-I, was enhanced in the presence of PTC-CM compared with control (1162.2+/-94.2 vs. 969.1+/-58.9 ng/mg protein/day, P < 0.05), but no change was observed in cortical fibroblast secretion of IGFBP-2 (260.9+/-38.8 vs. 290.9+/-36.6 ng/mg protein/day, P = NS) or IGF-I (56.7+/-6.6 vs. 57.0+/-6.8 ng/mg protein/day, P = NS). Human PTC secreted transforming growth factor-beta1 (TGF-beta1) and the AB heterodimer of platelet-derived growth factor (PDGF-AB) in a time-dependent fashion and the augmentation of cortical fibroblasts mitogenesis, collagen synthesis and IGFBP-3 secretion induced by PTC-CM was replicated by exogenous TGF-beta1 and PDGF. Furthermore, the stimulatory effects of PTC on cortical fibroblasts were potentiated in transiently acidified PTC-CM (which activated latent TGF-beta1), and were abrogated by neutralizing antibodies specifically directed against TGF-beta1 and PDGF-AB. Cortical fibroblasts in turn released a soluble factor(s) into cortical fibroblast-conditioned media that reciprocally stimulated PDGF-AB production by PTC (4.79+/-1.55 vs. 0.78+/-.06 ng/mg protein/day, P < 0.05). CONCLUSIONS: PTC modulate the biological behavior of neighboring cortical fibroblasts in the human kidney through paracrine mechanisms, which include the production and release of PDGF-AB and TGF-beta1. Renal insults that result in proximal tubule injury may perturb this paracrine interaction, thereby culminating in excessive fibroblast proliferation and interstitial fibrosis.