TGF-beta Activity In Conditioned Medium Research Articles

Differential Transcriptional Responses of Keloid and Normal Keratinocytes to Serum Stimulation

Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs. Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively. Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs. KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

IL-3 affects endothelial cell-mediated smooth muscle cell recruitment by increasing TGF beta activity: potential role in tumor vessel stabilization.

Interleukin-3 (IL-3) expression by tumor-infiltrating lymphocytes (TILs) and its effects on vessel assembly were evaluated. TILs from 'in situ' human breast cancers expressed CD4/CD25 antigens and IL-3. An injection of Matrigel containing SMC and IL-3 or basic-fibroblast growth factor (bFGF) into SCID mice confirmed the neoangiogenetic effect of both factors. However, in response to IL-3, but not to bFGF, only few SMC became incorporated into the nascent vessels. To evaluate the possibility that signals emanated by the nascent vasculature in the presence of IL-3 may negatively regulate SMC recruitment, conditioned media (CM) from IL-3-treated endothelial cells (EC) or SMC were tested for their biological effects on SMC and EC. CM from IL-3-treated SMC stimulated the migration of EC. In contrast, the migration of SMC was not affected by CM from IL-3-stimulated EC; however, it was greatly enhanced by blocking transforming growth factor beta (TGF beta) activity. TGF beta immunoenzymatic assay demonstrated the following: (i) the absence of TGF beta activity in CM from IL-3-stimulated EC; (ii) a barely detectable TGF beta activity in CM from IL-3-stimulated SMC; and (iii) the presence of TGF beta activity in the supernatants of SMC stimulated with CM from IL-3-, but not from bFGF-stimulated EC. Increased TGF beta mRNA expression was only detected in SMC stimulated with CM from IL-3-treated EC. Finally, the inhibitory signals induced by IL-3 in vivo were abrogated by the addition of the neutralizing TGF beta antibody. Thus, the positive immunostaining for IL-3 by TILs in 'in situ' breast cancers sustains the possibility that early in tumor development, IL-3 can contribute to the chronic immaturity of these vessels.

Osteogenic actions of phenytoin in human bone cells are mediated in part by TGF-beta 1.

We have recently demonstrated that phenytoin, a widely used therapeutic agent for seizure disorders, has osteogenic effects in rats and in humans in vivo, and in human bone cells in vitro. The goal of the present study was to determine the mechanism of the osteogenic action of phenytoin in normal human mandible-derived bone cells. Because many osteogenic agents increased bone cell proliferation through mediation by growth factors, we tested the hypothesis that the osteogenic effects of phenytoin involved the release of a growth factor by measuring the mRNA level of several bone cell growth factors and insulin-like growth factor (IGF) binding proteins with Northern blots using specific cDNA probes. Treatment with 5-50 microM phenytoin reproducibly and markedly increased (up to 6-fold, p < 0.001) the mRNA of transforming growth factor (TGF)-beta 1, but not that of other growth factors (i.e., IGF-II, platelet-derived growth factor-A [PDGF-A], PDGF-B, and TGF-beta 2) and IGF binding proteins (i.e., IGFBP-3, -4, and -5). The stimulation was dose dependent, with an optimal dose of 10-50 microM. Maximal increase was seen after 1 h of phenytoin treatment. The release of biologically active TGF-beta activity in conditioned media was measured with the mink lung cell proliferation inhibition assay. Twenty-four hours of phenytoin treatment significantly increased the production of biologically active TGF-beta (2-fold, p < 0.05) with the optimal dose between 5-50 microM. Comparisons between the in vitro osteogenic effects of phenytoin and those of TGF-beta 1 reveal that these two agents at their respective optimal doses had similar maximal stimulatory effects on [3H]thymidine incorporation, alkaline phosphatase (ALP)-specific activity, and type I alpha-2 collagen mRNA expression in human bone cells. The stimulatory effects of phenytoin on [3H]thymidine incorporation and ALP-specific activity were completely blocked by a neutralizing anti-TGF-beta antibody. In conclusion, these findings demonstrate for the first time that at least some of the osteogenic actions of phenytoin in human bone cells could be in part mediated by TGF-beta 1.

Tamoxifen decreases the rate of proliferation of rat vascular smooth-muscle cells in culture by inducing production of transforming growth factor beta.

Tamoxifen selectively and reversibly decreased the rate of proliferation of adult rat aortic vascular smooth-muscle cells (VSMCs). Half-maximal inhibition of proliferation occurred at 2-5 microM tamoxifen for VSMCs and at > 50 microM for adventitial fibroblasts. The cell cycle time for all the VSMCs in the population was increased from 35 +/- 2 h to 54 +/- 4 h in the presence of 33 microM tamoxifen. Tamoxifen did not affect the time of entry into DNA synthesis, but delayed arrival at mitosis by > 24 h. It therefore extended the duration of the G2-to-M phase of the cell cycle. However, the rate of proliferation of VSMCs was not decreased by tamoxifen (at concentrations up to 50 microM) in the presence of neutralizing antibody to transforming growth factor beta (TGF-beta). The level of mRNA for TGF-beta 1 in VSMCs was strongly induced by 10 microM tamoxifen, and TGF-beta activity in conditioned medium from tamoxifen-treated cells was more than 50-fold higher than from control cells. Tamoxifen therefore extended the G2-to-M phase of the cell cycle in VSMCs by increasing TGF-beta activity in the culture.

Angiotensin II-induced mitogenesis of spontaneously hypertensive rat-derived cultured smooth muscle cells is dependent on autocrine production of transforming growth factor-beta.

Angiotensin II (Ang II) has been implicated in the regulation of smooth muscle cell proliferation after vascular injury, but the molecular mechanisms of this effect remain obscure. The aims of the present study were 1) to determine if Ang II was mitogenic (in a defined serum-free medium) for aortic smooth muscle cells derived from spontaneously hypertensive rats, either alone or in combination with epidermal growth factor, basic fibroblast growth factor, or platelet-derived growth factor-BB; and 2) to determine if the Ang II effects were mediated by autocrine production of transforming growth factor-beta (TGF-beta). Results demonstrated that Ang II increased the proliferative response of smooth muscle cells to epidermal growth factor or platelet-derived growth factor-BB. Ang II alone and in combination with basic fibroblast growth factor induced a small delayed increase (48-72 hours after treatment) in DNA synthesis and [3H]thymidine labeling indexes without an increase in cell number. Ang II effects were at least partially mediated by autocrine production of active TGF-beta in that 1) treatment with Ang II increased TGF-beta activity in conditioned media and 2) TGF-beta neutralizing antibody inhibited Ang II-induced increases in DNA synthesis. However, treatment with exogenous TGF-beta at concentrations induced by Ang II failed to elicit a mitogenic response, thus implicating other autocrine factors in mediation of Ang II effects. Results suggest a potential mechanism whereby Ang II might regulate smooth muscle cell mitogenesis after vascular injury.

In vitro effects of bone- and platelet-derived transforming growth factor-? on the growth of Walker 256 carcinosarcoma cells

Conditioned media from fetal rat calvarial cultures has previously been shown to stimulate the growth of the bone-metastasizing Walker 256 carcinosarcoma cell line. In the current investigation we looked at the possibility that transforming growth factor-beta (TGF-beta), present in conditioned media, and positively correlated with resorption in vitro, may be responsible for the enhanced proliferation of Walker cells cultured in these conditioned media. Purified platelet-derived TGF-beta produced a dose-dependent growth response in Walker cells with an ED50 equal to 0.05 ng/ml. Bone-derived TGF-beta activity in conditioned media, measured by NRK fibroblast colony formation, correlated well with percentage resorption in bone cultures, and growth activity in Walker cell culture. In addition to this, the growth response normally seen with conditioned media cultures of Walker cells was significantly inhibited by the addition of anti-TGF-beta 1 neutralizing antibody. We conclude that TGF-beta is an important growth stimulatory component from fetal rat calvaria.