Persistence of TGF-β1 induction of increased fibroblast contractility

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.     

The single-molecule mechanics of the latent TGF-β1 complex

Background

TGF-β1 controls many pathophysiological processes including tissue homeostasis, fibrosis, and cancer progression. Together with its latency-associated peptide (LAP), TGF-β1 binds to the latent TGF-β1-binding protein-1 (LTBP-1), which is part of the extracellular matrix (ECM). Transmission of cell force via integrins is one major mechanism to activate latent TGF-β1 from ECM stores. Latent TGF-β1 mechanical activation is more efficient with higher cell forces and ECM stiffening. However, little is known about the molecular events involved in this mechanical activation mechanism.

Results

By using single-molecule force spectroscopy and magnetic microbeads, we analyzed how forces exerted on the LAP lead to conformational changes in the latent complex that can ultimately result in TGF-β1 release. We demonstrate the unfolding of two LAP key domains for mechanical TGF-β1 activation: the α1 helix and the latency lasso, which together have been referred to as the “straitjacket” that keeps TGF-β1 associated with LAP. The simultaneous unfolding of both domains, leading to full opening of the straitjacket at a force of ∼40 pN, was achieved only when TGF-β1 was bound to the LTBP-1 in the ECM.

Conclusions

Our results directly demonstrate opening of the TGF-β1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-β1 to LTBP-1 is mandatory. Interfering with mechanical activation of latent TGF-β1 by reducing integrin affinity, cell contractility, and binding of latent TGF-β1 to the ECM provides new possibilities to therapeutically modulate TGF-β1 actions.     

TGF-β1受体1和2在TGF-β1调节细胞增殖中的作用

转化生长因子β1(transforming growth factor-β1,TGF-β1)是一种多功能细胞因子,在细胞增殖、分化、伤口愈合和肿瘤生成转移等过程中均发挥重要调控作用。TGF-β1对细胞增殖的调节可因细胞类型、刺激剂量不同而不同,但其差异调节的机制还不清楚。现普遍认为,TGF-β1在TGFBR2/TGFBR1二聚体参与下通过经典的Smad信号通路抑制增殖,而通过非Smad信号通路促进细胞周期,但是机体是如何调控这种不同增殖调节作用转化的还不明确。TGFBR1和TGFBR2在细胞中的分布和比例变化可能是TGF-β1差异性调控细胞增殖作用的一个重要机制。     

Influence of TGF-β1 on the expression of BSP,DSP, TGF-β1 receptor I and Smad proteins during reparative dentinogenesis

Reparative dentin has a wide variety of manifestations ranging from a regular, tubular form to an irregular, atubular form. However, the characteristics of reparative dentin have not been clarified. This study hypothesized that the level of bone sialoprotein (BSP) expression will increase if the newly formed reparative dentin is bone-like but the dentin sialophosphoprotein (DSPP) level will decrease. In order to test this hypothesis, the expression of BSP and DSP was examined by immunohistochemistry and the expression of BSP was measured by in situ hybridization in an animal model. The pulps of 12 maxillary right first molars from twelve male rats were exposed and capped with MTA. In addition, in order to understand the role of transforming growth factor-beta 1 (TGF-β1) during reparative dentinogenesis, the expression of BSP and DSPP mRNA was analyzed by RT-PCR in a human dental pulp cell culture, and the transforming growth factor-beta 1 receptors (TβRI) and Smad 2/3 were examined by immunofluorescence in an animal model. DSP was expressed in the normal odontoblasts and odontoblast-like cells of the reparative dentin. Interestingly, BSP was strongly expressed in the odontoblast-like cells of reparative dentin. The level of the TβRI and Smad 2/3 proteins was higher in the reparative dentin than in the normal dentin. TGF-β1 up-regulated BSP in the human pulp cell cultures. This suggests that reparative dentin has both dentinogenic and osteogenic characteristics that are mediated by TGF-β1.     

Separation, purification, and sequence identification of TGF-β1 and TGF-β2 from bovine milk

Cox and Bürk (Eur. J. Biochem., 1991) reported the partial characterization of Milk Growth Factor (MGF) which stimulated the migration of fibroblasts. We have fractionated the partially purified sample by RP-HPLC and obtained the separation of two peaks of activity. The two active components were isolated as pure MGF-a and MGF-b by RP-HPLC and preparative SDS-PAGE. The purified MGF-a, consisting of a single band by gel electrophoresis and a single peak on an HPLC reversed-phase C-4 column, has the same specific activity as TGF-2 in the fibroblast migration assay. MGF-a was digested by endoprotease Asp-N and the cleaved peptides were analyzed by Edman degradation and plasma desorption mass spectrometry (PDMS). The whole sequence of MGF-a determined by automated sequenator and PDMS of S-pyridylethylated protein and selected fragments was found to be identical to that of TGF-2. MGF-b protein mixture separated by SDS-PAGE was electrophoretically transferred onto a Biometra Glassybond membrane, and the blotted MGF-b protein was directly sequenced on an automated sequenator. The identified 29 amino acids sequence of MGF-b was identical to the amino-terminal sequence of TGF-1. Our study demonstrates that MGF is composed of both TGF-1 and TGF-2. TGF-2 (85%) is the predominant form.     

β3 Integrin Promotes TGF-β1/H2O2/HOCl-Mediated Induction of Metastatic Phenotype of Hepatocellular Carcinoma Cells by Enhancing TGF-β1 Signaling

In addition to being an important mediator of migration and invasion of tumor cells, β3 integrin can also enhance TGF-β1 signaling. However, it is not known whether β3 might influence the induction of metastatic phenotype of tumor cells, especially non-metastatic tumor cells which express low level of β3. Here we report that H₂O₂ and HOCl, the reactive oxygen species produced by neutrophils, could cooperate with TGF-β1 to induce metastatic phenotype of non-metastatic hepatocellular carcinoma (HCC) cells. TGF-β1/H₂O₂/HOCl, but not TGF-β1 or H₂O₂/HOCl, induced β3 expression by triggering the enhanced activation of p38 MAPK. Intriguingly, β3 in turn promoted TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of HCC cells by enhancing TGF-β1 signaling. β3 promoted TGF-β1/H₂O₂/HOCl-induced expression of itself via positive feed-back effect on p38 MAPK activation, and also promoted TGF-β1/H₂O₂/HOCl-induced expression of α3 and SNAI2 by enhancing the activation of ERK pathway, thus resulting in higher invasive capacity of HCC cells. By enhancing MAPK activation, β3 enabled TGF-β1 to augment the promoting effect of H₂O₂/HOCl on anoikis-resistance of HCC cells. TGF-β1/H₂O₂/HOCl-induced metastatic phenotype was sufficient for HCC cells to extravasate from circulation and form metastatic foci in an experimental metastasis model in nude mice. Inhibiting the function of β3 could suppress or abrogate the promoting effects of TGF-β1/H₂O₂/HOCl on invasive capacity, anoikis-resistance, and extravasation of HCC cells. These results suggest that β3 could function as a modulator to promote TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of non-metastatic tumor cells, and that targeting β3 might be a potential approach in preventing the induction of metastatic phenotype of non-metastatic tumor cells.     

Sustained anti-inflammatory effects of TGF-β1 on microglia/macrophages

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3 days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7 dpr when transforming growth factor-β1 (TGF-β1) expression was robustly increased. After transient incubation with TGF-β1 for 24 h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72 h after removal of TGF-β1. The sustained TGF-β1 effects were not attributable to microglia-derived endogenous TGF-β1, as revealed by TGF-β1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-β1 receptor-selective blocker SB525334. After incubation with TGF-β1 for 24 h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-β1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3 dpr, whereas it was almost disappeared on 7 dpr. The findings suggest that abundantly produced TGF-β1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.     

The Interaction of Endothelin-1 and TGF-β1 Mediates Vascular Cell Remodeling

Background

Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor.

Objective

To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC.

Methods

PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580.

Results

ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27^(Kip). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1.

Conclusion and Clinical Relevance

ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.     

TGF-β1 Induced Transdifferentiation of RPE Cells is Mediated by TAK1

Background and Aim

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.

Methodology

ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.

Results

This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.

Conclusions/Significance

This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment.     

Dynamics of TGF-β/Smad signaling

The physiological responses to TGF-β stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the non-canonical TGF-β signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-β signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-β biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-β signaling and how cells quantitatively decode variable doses of TGF-β stimulation.     

Role of tumor-derived transforming growth factor-β1 (TGF-β1) in site-dependent tumorigenicity of murine ascitic lymphosarcoma

An ascitic lymphosarcoma (LS-A) of Swiss mice that regressed spontaneously on subcutaneous (s.c.) transplantation was investigated for the mechanism of its progressive growth and host mortality on intraperitoneal (i.p.) transplantation. In vitro studies indicated significant inhibition of LS-A proliferation seeded at higher cell density (>10⁴/ml). Culture supernatants of LS-A caused bi-modal growth effects, the early supernatants (24 h) caused stimulation and the late (72 h) supernatants inhibited LS-A proliferation. The 72-h supernatants also suppressed T and B cell response to mitogens in a dose-dependent manner. Pan anti-transforming growth factor- antibody abrogated the inhibitory effects of supernatants. The supernatants contained both latent as well as bio-active form of transforming growth factor-1 (TGF-1) as determined by ELISA. Mice bearing i.p. ascites tumor had elevated serum TGF-1, hemoglobulinemia, splenic lymphopenia, impaired response of the T cells to mitogen and reduced expression of transferrin receptor (CD71) on the bone marrow cells. However, mice which rejected s.c. transplants, did not show significant changes in these parameters. Our studies indicated profound influence of site of tumor growth on tumor progression and host immune system mediated by tumor-derived TGF-1. It is possible that human tumors which secrete TGF-1 may exhibit similar patho-physiological effects in the host depending on the anatomical site of the tumor.     

SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-β1 activity

Pericytes migrate to nascent vessels and promote vessel stability. Recently, we reported that secreted protein acidic and rich in cysteine (SPARC)-deficient mice exhibited decreased pericyte-associated vessels in an orthotopic model of pancreatic cancer, suggesting that SPARC influences pericyte behavior. In this paper, we report that SPARC promotes pericyte migration by regulating the function of endoglin, a TGF-β1 accessory receptor. Primary SPARC-deficient pericytes exhibited increased basal TGF-β1 activity and decreased cell migration, an effect blocked by inhibiting TGF-β1. Furthermore, TGF-β-mediated inhibition of pericyte migration was dependent on endoglin and αV integrin. SPARC interacted directly with endoglin and reduced endoglin interaction with αV integrin. SPARC deficiency resulted in endoglin-mediated blockade of pericyte migration, aberrant association of endoglin in focal complexes, an increase in αV integrins present in endoglin immunoprecipitates, and enhanced αV integrin-mediated activation of TGF-β. These results demonstrate that SPARC promotes pericyte migration by diminishing TGF-β activity and identify a novel function for endoglin in controlling pericyte behavior.     

TGF-β1 Protects against Mesangial Cell Apoptosis via Induction of Autophagy

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-β1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-β1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-β1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3^−/−) mice. We show that TGF-β1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-β1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-β1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-β1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.     

TGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells

Background

Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E₂) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.

Methodology/Principal Findings

Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E₂ as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E₂ secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E₂ and TGF-β1, and E₂ treatment in turn restored the inhibition of TGF-β1 on GJIC.

Conclusions

Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E₂ secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.     

Induction of galectin-1 by TGF-β1 accelerates fibrosis through enhancing nuclear retention of Smad2

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.     

FOXO1 promotes wound healing through the up-regulation of TGF-β1 and prevention of oxidative stress

Keratinocyte mobilization is a critical aspect of wound re-epithelialization, but the mechanisms that control its precise regulation remain poorly understood. We set out to test the hypothesis that forkhead box O1 (FOXO1) has a negative effect on healing because of its capacity to inhibit proliferation and promote apoptosis. Contrary to expectations, FOXO1 is required for keratinocyte transition to a wound-healing phenotype that involves increased migration and up-regulation of transforming growth factor β1 (TGF-β1) and its downstream targets, integrin-α3 and -β6 and MMP-3 and -9. Furthermore, we show that FOXO1 functions in keratinocytes to reduce oxidative stress, which is necessary to maintain cell migration and prevent cell death in a TGF-β1–independent manner. Thus, our studies identify a novel function for FOXO1 in coordinating the response of keratinocytes to wounding through up-regulation of TGF-β1 and other factors needed for keratinocyte migration and protection against oxidative stress, which together promote migration and decrease apoptosis.     

Short communication: Concentration of TGF-β1, IL-10 and IL-6 in boar seminal plasma and TGF-β1 level in different fractions of ejaculates

Levels of the cytokines transforming growth factor (TGF)-β1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-β1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-β1 and IL-10) and pro-inflammatory (IL-6). Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-β1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF β1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10 ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-β1 evaluation. In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-β1 (20.4 - 766.5 pg/mL) and IL-10, (73.7 - 837.3 pg/mL), was found. For IL-6, the concentration was low (range 11.5 - 30.9 pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-β1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-β1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-β1 was found in the 10SRF than in the other fractions. Different storage temperatures (-20°C or -80°C) did not affect the seminal plasma TGF-β1 level after one year of storage. To conclude: Boar seminal plasma contained TGF- β1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- β1 level was highest in the first 10 mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- β1.