Presence Of TGF-beta Research Articles

Erk1/2 signaling is required for Tgf‐β2–induced suture closure

Transforming growth factors beta (Tgf-betas) act by means of Smad signaling pathways and may also interact with the mitogen-activated protein kinase pathway. The hypothesis was tested that Erk1/2 signaling is required for Tgf-beta2-induced suture closure, by culturing embryonic mouse calvariae in the presence of Tgf-beta2 with or without Erk1/2 inhibitor PD98059 (PD). Suture widths were measured daily, and microdissected sutures and bones were homogenized and protein analyzed by Western blots. Tgf-beta2 induced narrowing of the sutures after 72 hr, an effect inhibited by treatment with PD. Erk1/2 and Egf but not Smad2/3 protein expression was up-regulated by Tgf-beta2 calvarial tissues at 72 hr. PD inhibited endogenous and Tgf-beta2-stimulated Erk1/2 protein as well as Tgf-beta2-stimulated Egf, but increased Smad2/3 protein expression. In tissues harvested 0, 15, and 30 min after exposure to Tgf-beta2, Erk1/2 phosphorylation was up-regulated after 15 min, an effect abrogated by the simultaneous addition of PD. In summary, Tgf-beta2 stimulated Erk1/2 phosphorylation and induced Egf and Erk1/2 expression, associated with suture closure after 72 hr. Blocking Erk1/2 activity with PD inhibited these effects but increased Smad2/3 expression. We postulate that Tgf-beta2 regulates suture closure directly by means of phosphorylation of Erk1/2 and indirectly by up-regulating Erk1/2, a substrate for Fgf receptor signaling required for Fgf induction of premature suture obliteration.

Transforming growth factor‐β3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk‐1, and CD31 gene expression during CL/Fr mouse lip fusion

Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.

The Heterogeneous Murine Corneal Stromal Cell Populations In Vitro

To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

TGF-β 1 inhibits late-stage mast cell maturation

The objective of this study was to determine the effects of transforming growth factor (TGF)-beta1 on mast cell development. Mast cells were cultured from mouse bone marrow in interleukin (IL)-3 + stem cell factor, in the presence or absence of TGF-beta1. We assessed mast cell development by measuring the expression of kit, T1/ST2, FcvarepsilonRI, and Fcgamma receptors. Cell morphology was determined by histochemical staining. Alterations in FcvarepsilonRI subunit expression were measured by Western blot analysis. Adoptive transfer of cultured mast cells into mast cell-deficient W/W(v) mice was used to determine if the in vivo environment could reverse the inhibitory effects of TGF-beta1. TGF-beta1 decreased FcvarepsilonRI, c-kit, T1/ST2, and FcgammaR expression, and inhibited granule formation in developing mast cells. Accessory cells were not required for this inhibition. Smad3 deficiency did not alter the response of bone marrow cells to TGF-beta1. TGF-beta1 inhibited expression of the FcvarepsilonRI alpha subunit protein, without decreasing beta or gamma proteins. Mast cells derived in the presence of TGF-beta1 were functionally impaired, as IgE-mediated cytokine secretion was greatly reduced. The changes in granule formation and surface antigen expression were long-standing, as they were not reversed by transfer to W/W(v) mice. TGF-beta1 may contribute to mast cell homeostasis by inhibiting maturation from bone marrow precursors. The effects of TGF-beta1 result in greatly diminished expression of cell surface markers, reduced granulation, and lack of responsiveness to IgE-mediated activation. Thus TGF-beta1 can serve as a potent and multifunctional regulator of mast cell maturation.

Regulation of mesenchymal stem cell and chondrocyte differentiation by MIA

Melanoma inhibitory activity (MIA), also referred to as cartilage-derived retinoic acid-sensitive protein (CD-RAP), an 11-kDa secreted protein, is mainly expressed in cartilaginous tissue during embryogenesis and adulthood. Currently, the function of MIA in cartilage tissue is not understood. Here, we describe that MIA acts as a chemotactic factor on the mesenchymal stem cell line C3H10T1/2, stimulating cell migration significantly at concentrations from 0.24 to 240 ng/ml, while inhibiting cell migration at higher doses of 2.4 microg/ml. When analyzing the role of MIA during differentiation processes, we show that MIA by itself is not capable to induce the differentiation of murine or human mesenchymal stem cells. However, MIA influences the action of bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta 3 during mesenchymal stem cell differentiation, supporting the chondrogenic phenotype while inhibiting osteogenic differentiation. Quantitative RT-PCR analysis revealed the up-regulation of the cartilage markers MIA, collagen type II and aggrecan in human mesenchymal stem cell (HMSC) cultures differentiated in the presence of MIA and TGF-beta 3 or BMP-2 when compared to HMSC cultures differentiated in the presence of TGF-beta 3 or BMP-2 alone. Further, MIA down-regulates gene expression of osteopontin and osteocalcin in BMP-2 treated HMSC cultures inhibiting the osteogenic potential of BMP-2. In the case of human primary chondrocytes MIA stimulates extracellular matrix deposition, increasing the glycosaminoglycan content. Therefore, we postulate that MIA is an important regulator during chondrogenic differentiation and maintenance of cartilage.

Somatic Acquisition and Signaling of <EMPH TYPE="ITAL">TGFBR1</EMPH>*6A in Cancer

TGFBR1*6A is a common polymorphism of the type I transforming growth factor beta receptor (TGFBR1). Epidemiological studies suggest that TGFBR1*6A may act as a tumor susceptibility allele. How TGFBR1*6A contributes to cancer development is largely unknown. To determine whether TGFBR1*6A is somatically acquired by primary tumors and metastases during cancer development and whether the 3-amino acid deletion that differentiates TGFBR1*6A from TGFBR1 is part of the mature receptor or part of the signal sequence and to investigate TGFBR1*6A signaling in cancer cells. Tumor and germline tissues from 531 patients with a diagnosis of head and neck, colorectal, or breast cancer recruited from 3 centers in the United States and from 1 center in Spain from June 1, 1994, through June 30, 2004. In vitro translation assays, MCF-7 breast cancer cells stably transfected with TGFBR1*6A, TGFBR1, or the vector alone, DLD-1 colorectal cancer cells that endogenously carry TGFBR1*6A, and SW48 colorectal cancer cells that do not carry TGFBR1*6A. TGFBR1*6A somatic acquisition in cancer. Determination of the amino terminus of the mature TGFBR1*6A and TGFBR1 receptors. Determination of TGF-beta-dependent cell proliferation. TGFBR1*6A was somatically acquired in 13 of 44 (29.5%) colorectal cancer metastases, in 4 of 157 (2.5%) of colorectal tumors, in 4 of 226 (1.8%) head and neck primary tumors, and in none of the 104 patients with breast cancer. TGFBR1*6A somatic acquisition is not associated with loss of heterozygosity, microsatellite instability, or a mutator phenotype. The signal sequences of TGFBR1 and TGFBR1*6A are cleaved at the same site resulting in identical mature receptors. TGFBR1*6A may switch TGF-beta growth inhibitory signals into growth stimulatory signals in MCF-7 breast cancer cells and in DLD-1 colorectal cancer cells. TGFBR1*6A is somatically acquired in 29.5% of liver metastases from colorectal cancer and may bestow cancer cells with a growth advantage in the presence of TGF-beta. The functional consequences of this conversion appear to be mediated by the TGFBR1*6A signal sequence rather than by the mature receptor. The results highlight a new facet of TGF-beta signaling in cancer and suggest that TGFBR1*6A may represent a potential therapeutic target in cancer.

Transforming growth factor β stimulates rheumatoid synovial fibroblasts via the type II receptor

Transforming growth factor (TGF)-beta regulates the function of fibroblasts, and has been shown to have a role in the pathogenesis of rheumatoid arthritis (RA) because several studies have demonstrated the presence of TGF-beta in the synovial tissue and synovial fluids of RA patients. In this study, we examined the expression of TGF-beta receptors in synovial fibroblasts of patients with RA and demonstrated the significance in functional responses of synovial fibroblasts to TGF-beta in this disorder. Transforming growth factor beta1 stimulated the expression of connective tissue growth factor (CTGF) in fibroblasts of patients with RA more than in those of patients with osteoarthritis (OA). Transforming growth factor beta1 induced the chemotactic migration of RA synovial fibroblasts and inhibited their proliferation significantly more than OA synovial fibroblasts. Both RA and OA synovial fibroblasts expressed detectable amounts of TGF-beta receptor type II mRNA, but the expression was higher in RA patients than in OA patients, as assessed by reverse transcriptase-polymerase chain reaction. There was no significant difference in the expression of TGF-beta receptor type I or type III in synovial fibroblasts between RA and OA patients. These results indicate that synovial fibroblasts of RA patients express the increased TGF-beta receptor type II, which is associated with altered responses to TGF-beta observed in CTGF expression, chemotaxis, and proliferation of RA synovial fibroblasts, and may have an important role in the pathogenesis of RA.

Induction of Intervertebral Disc–Like Cells From Adult Mesenchymal Stem Cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.

The Smad3 linker region contains a transcriptional activation domain.

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

TGF-beta impairs renal autoregulation via generation of ROS.

Impaired autoregulation in chronic kidney disease can result in elevation of glomerular capillary pressure and progressive glomerular damage; however, the factors linking chronic glomerular disorders to impaired autoregulation have not been identified. We tested the hypothesis that the cytokine most closely associated with progressive glomerular disease, transforming growth factor (TGF)-beta, may also attenuate autoregulation. Kidneys from normal rats were prepared for videomicroscopy, using the blood-perfused juxtamedullary nephron technique. Autoregulatory responses were measured under control conditions and during superfusion with TGF-beta1 (10 ng/ml). Control afferent arteriolar diameter averaged 18.4 +/- 1 microm and significantly decreased to 16.3 +/- 0.9 and 13.2 +/- 0.8 microm at perfusion pressures of 130 and 160 mmHg, respectively. In the presence of TGF-beta1, autoregulatory responses were completely blocked. In similar experiments performed using PDGF-BB (10 ng/ml) and HGF (25 ng/ml), the normal autoregulatory response was not affected. In vitro studies, using isolated preglomerular vascular smooth muscle cells, revealed that exposure to TGF-beta1 stimulated a rapid increase in reactive oxygen species (ROS) that was inhibited by NADPH oxidase inhibitors. In situ studies, with dihydroethidium staining, revealed a marked increase in renal vessel ROS production on exposure to TGF-beta1. Pretreatment of the juxtamedullary afferent arterioles with tempol, a ROS scavenger, or with apocynin, a NADPH oxidase inhibitor, prevented the impaired autoregulation induced by TGF-beta1. These data reveal a novel hemodynamic pathway by which TGF-beta could lead to progressive glomerular injury by impairing normal renal microvascular function.

Regulation of the expression of 5‐lipoxygenase–activating protein/5‐lipoxygenase and the synthesis of leukotriene B4 in osteoarthritic chondrocytes: Role of transforming growth factor β and eicosanoids

To explore the modulation of 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX) expression in human osteoarthritic (OA) chondrocytes, their relative implications in leukotriene B(4) (LTB(4)) production, the effect of different factors on this system, and the influence of increased LTB(4) production on the synthesis of catabolic factors of cartilage. FLAP and 5-LOX expression and LTB(4) production were monitored following treatment with transforming growth factor beta1 (TGFbeta1; 5 ng/ml) and 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3); 50 nM) alone or in combination with selective or nonselective cyclooxygenase (COX) inhibitors, naproxen (90 mug/ml), NS-398 (10 muM), or FR122047 (5 muM), or a dual inhibitor of COX/5-LOX activity, licofelone (2.6 muM). LTB(4), prostaglandin E(2) (PGE(2)), and matrix metalloprotease 1 (MMP-1) production were measured by specific enzyme-linked immunosorbent assays, nitric oxide by the Griess reaction, and FLAP and 5-LOX expression by quantitative polymerase chain reaction. Human OA chondrocytes expressed both FLAP and 5-LOX. TGFbeta1 and/or 1,25(OH)(2)D(3) induced a rapid and marked enhancement ( approximately 4-13-fold) in FLAP messenger RNA (mRNA) levels, which was associated with a subsequent and late increase in LTB(4) production and PGE(2) synthesis. Treatment with COX inhibitors in the absence or presence of TGFbeta1 and 1,25(OH)(2)D(3) induced a rapid increase in LTB(4) production; this response was mediated by the sustained and significant (P < 0.01) up-regulation ( approximately 1.5-fold) of 5-LOX mRNA levels. Conversely, treatment with licofelone showed no effect on 5-LOX but significantly reduced FLAP expression levels. Coincubation of licofelone with TGFbeta1 plus 1,25(OH)(2)D(3) did not affect FLAP or 5-LOX levels. In the presence of TGFbeta1 plus 1,25(OH)(2)D(3), naproxen, but not licofelone, induced MMP-1 production and both drugs decreased nitric oxide levels. Both the eicosanoids PGE(2) and LTB(4) are important cofactors in regulating FLAP/5-LOX expression; the inhibition of PGE(2) up-regulates 5-LOX while down-regulating FLAP gene expression, and LTB(4) appears to be an up-regulating factor on the 5-LOX gene. Importantly, nonsteroidal antiinflammatory drugs up-regulate the synthesis of LTB(4), supporting the shunt hypothesis from COX to 5-LOX. We also demonstrated that LTB(4) likely contributes to the up-regulation of important catabolic factors involved in the pathophysiology of OA, such as MMP.

Hypoxia and Transforming Growth Factor-β1 Act Independently to Increase Extracellular Matrix Production by Placental Fibroblasts

Villous fibrosis is associated with oxygen deprivation in placental pathology, but the signaling networks and growth factors involved in activating the relevant cellular repair mechanisms are largely unknown. TGF is a powerful enhancer of extracellular matrix (ECM) production and an important immune suppressor that has been linked with fibrosis in several tissues. Here, cell culture methods were used to investigate possible links between hypoxia, elevated TGF beta 1, and altered ECM production in placenta. Term placental fibroblasts were isolated and cultured under hypoxia (3% O(2)) or in the presence of TGF beta 1, and the expression of fibronectin, collagen I, and collagen IV was examined using immunohistochemistry, ELISA of cell monolayers with associated ECM, and real-time RT-PCR. The effect of hypoxia on endogenous production of TGF beta 1-3 was also examined. Both TGF beta 1 and hypoxia increased fibronectin, collagen I, and collagen IV protein and mRNA in placental fibroblasts. However, TGF beta 1-3 production was not increased by culturing the cells under hypoxic conditions for 5 d. Thus, increased ECM expression under hypoxia was not mediated directly by increased TGF beta. We conclude that ECM production can be stimulated independently by hypoxia and TGF beta 1.

Collagen, type V, α1 (COL5A1) is regulated by TGF-β in osteoblasts

Bone matrix contains high concentrations of growth factors that are known to play important regulatory roles during osteogenesis, particularly transforming growth factor-beta (TGF-beta). Divergent effects of TGF-beta on bone formation have been reported both in vitro and in vivo depending upon experimental conditions, cells employed and their stage of maturation. In this study, we have used a clonal osteoblastic cell line MC3T3-E1, derived from newborn mouse calvaria, as an in vitro model of bone development. These cells undergo an ordered, time-dependent developmental sequence characterized by three stages (proliferation, differentiation and mineralization), over a 30-35-day period. In this study, cDNA microarray technology was used to study the expression profile of 8470 genes, in the presence of TGF-beta1 during osteoblast development. Microarray analysis revealed 120 cDNAs to be differentially expressed in MC3T3-E1 osteoblasts that had been treated with TGF-beta1. From the 120 differentially expressed genes, we selected Collagen, type V, alpha1 (COL5A1) {differential expression=+4.9} for further studies since it represents a previously uncharacterized component of the bone matrix. Using Northern blotting, we found that, when MC3T3-E1 cells were treated with TGF-beta1, COL5A1 was up-regulated during the proliferation and differentiation phases of osteogenesis. Furthermore, by a combination of RNA in situ hybridization and Northern blotting, we found COL5A1 mRNA to be expressed in the calvaria and developing bone of the E17.5 mouse embryos. Lastly, significant COL5A1 protein expression was observed by immunohistochemistry in the developing bone of the E17.5 mouse embryos. In conclusion, by the use of in vitro and in vivo approaches, we have discovered that the COL5A1 gene is a target of TGF-beta during osteogenesis.

P21Cip1 and p27Kip1 act in synergy to alter the sensitivity of naive T cells to TGF-beta-mediated G1 arrest through modulation of IL-2 responsiveness.

Induction of G(1) arrest by TGF-beta correlates with the regulation of p21(Cip1) and p27(Kip1), members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-beta(1)-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-beta is diminished in T cells from mice deficient for both p21(Cip1) and p27(Kip1) (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8(+) T cells from DKO mice, TGF-beta is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15(Ink4b) in T cells stimulated in the presence of TGF-beta is not essential, as TGF-beta also efficiently suppressed proliferation of T cells from p15(Ink4b-/-) mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-beta, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3(-/-) T cells to the induction of growth arrest by TGF-beta. In summary, the growth suppressive effects of TGF-beta in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-beta by lowering thresholds for a maximal mitogenic response.

Transforming growth factor-beta-induced stimulation of formation of collagen fiber network and anti-fibrotic effect of taurine in an in vitro model of hepatic fibrosis.

The cell strain M, which was established from normal rat liver cells, is characterized by the active formation of a collagen fiber network. In this study, we investigated the characterization of M cells and evaluated the anti-fibrogenic effects of taurine using this culture system. M cells expressed cytokeratin (CK)8, CK18, vimentin, and alpha-smooth muscle actin, whereas expression of CK-19 or desmin was not detected. Also, M cells expressed transforming growth factor (TGF)-beta1, -beta2, and TGF-beta type I and II receptors, and treatment with TGF-beta1 (1ng/ml) for 6 days markedly stimulated the formation of a collagen fiber network and expression of procollagen alpha1(I) mRNA. When M cells were treated with various concentrations of taurine (10-50mM), network formation and procollagen alpha1(I) expression were significantly suppressed in a dose dependent manner. Additionally, even in the presence of TGF-beta1, taurine treatment effectively reduced the formation of a collagen fiber network. These results suggest that M cells exhibit features of not only hepatocytes but also myofibroblasts, and TGF-beta1 plays an important role in the formation of collagen fiber networks in this culture system. Additionally, this M cell culture system is appropriate for use as an in vitro model of hepatic fibrosis in the evaluation of the anti-fibrogenic effects of various agents.

Relaxin modulates cardiac fibroblast proliferation, differentiation, and collagen production and reverses cardiac fibrosis in vivo.

Cardiac fibrosis is a key component of heart disease and involves the proliferation and differentiation of matrix-producing fibroblasts. The effects of an antifibrotic peptide hormone, relaxin, in inhibiting this process were investigated. We used rat atrial and ventricular fibroblasts, which respond to profibrotic stimuli and express the relaxin receptor (LGR7), in addition to two in vivo models of cardiac fibrosis. Cardiac fibroblasts, when plated at low density or stimulated with TGF-beta or angiotensin II (Ang II), accelerated fibroblast differentiation into myofibroblasts, as demonstrated by significantly increased alpha-smooth muscle actin expression, collagen synthesis, and collagen deposition (by up to 95% with TGF-beta and 40% with Ang II; all P < 0.05). Fibroblast proliferation was significantly increased by 10(-8) m and 10(-7) m Ang II (63-75%; P < 0.01) or 0.1-1 microg/ml IGF-I (27-40%; P < 0.05). Relaxin alone had no marked effect on these parameters, but it significantly inhibited Ang II- and IGF-I-mediated fibroblast proliferation (by 15-50%) and Ang II- and TGF-beta-mediated fibroblast differentiation, as detected by decreased expression of alpha-smooth muscle actin (by 65-88%) and collagen (by 60-80%). Relaxin also increased matrix metalloproteinase-2 expression in the presence of TGF-beta (P < 0.01) and Ang II (P < 0.05). Furthermore, relaxin decreased collagen overexpression when administered to two models of established fibrotic cardiomyopathy, one due to relaxin deficiency (by 40%; P < 0.05) and the other to cardiac-restricted overexpression of beta2-adrenergic receptors (by 58%; P < 0.01). These coherent findings indicate that relaxin regulates fibroblast proliferation, differentiation, and collagen deposition and may have therapeutic potential in diseased states characterized by cardiac fibrosis.

Activation of Epstein–Barr virus by saliva from Sjogren's syndrome patients

The aim of this study was to examine the mechanism of Epstein-Barr virus (EBV) activation by soluble factors from the inflamed salivary glands of patients with Sjogren's syndrome (SS). Saliva from SS patients was used to examine the regulation of EBV activation by an inflammatory salivary microenvironment. Transient transfection of the EBV-negative salivary gland cell line (HSY) with BZLF1, a trans-activating EBV gene promoter-fusion construct (Zp-luc), was used in this study. The results showed that under conditions where the BZLF1 promoter is activated by potent stimuli, SS saliva (from eight of 12 patients) exerts a significant effect on expression of the luciferase gene. A specific inhibitor of protein kinase C did not affect the SS saliva-induced Zp-luc activity, whereas treatment with inhibitors of calmodulin, calcineurin and IP3, dose-dependently decreased this induction. Transforming growth factor beta1 (TGF-beta1), which is known to be expressed in SS salivary glands, dose-dependently induced Zp-luc activity. Hence, these results demonstrate the activation of EBV by SS saliva and suggest that EBV activation at the inflammatory site may occur in the presence of TGF-beta1 via triggering of the mitogen-activated protein kinase (MAPK) kinase signalling pathway.

Decrease of Smad4 gene expression in patients with essential thrombocythaemia may cause an escape from suppression of megakaryopoiesis by transforming growth factor-beta1.

Essential thrombocythaemia (ET) is characterized by the abnormal and sustained proliferation of megakaryocytes. The mechanism for this lineage-specific expansion in ET, remains unclear. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is involved in negative feedback regulation of megakaryopoiesis in both healthy volunteers (HV) and patients with idiopathic thrombocytopenic purpura (ITP). The present study found that megakaryocyte colony-forming units (CFU-MK) of ET patients were less sensitive to TGF-beta1 than those of HV. The expression of Smad4 (Sma- and Mad-related protein-4) in CFU-MK of ET patients was reduced in comparison with that of HV. Finally, to confirm that the impaired TGF-beta1 sensitivity was caused by reduced expression of Smad4, we examined Smad4-transfected CFU-MK from ET patients in the presence of TGF-beta1, and verified that the transfectants were indeed as susceptible as CFU-MK from HV to TGF-beta1. Thus it was surmised that one of the mechanisms for impaired sensitivity of CFU-MK to TGF-beta1 is the reduced expression of Smad4.

Negative regulation of SCFSkp2 ubiquitin ligase by TGF-beta signaling.

TGF-beta is a multifunctional growth factor whose best-known function is to inhibit cell growth and suppress tumor formation. TGF-beta causes cells to accumulate in mid-to-late G1 phase by blocking the transition from G1 to S. It has been shown that TGF-beta inhibits Cdk2-cyclin E kinase activity by promoting the binding of cell cycle inhibitor p27Kip1 to the kinase complexes. Here, we show that TGF-beta treatment leads to stabilization of p27Kip1 during G1 to S transition. We found that TGF-beta negatively regulates components of the SCF complex, which degrades the p27Kip1 during the G1 to S transition, through two distinct mechanisms. Using a pulse-chase analysis, we demonstrated that the stability of Skp2 decreases in the presence of TGF-beta. Destabilization of Skp2 by ubiquitin-mediated proteolysis was also demonstrated that in an in vitro degradation system, using cell extracts prepared from TGF-beta-treated cultured cells. In addition, TGF-beta treatment decreases the levels of Cks1 mRNA. The deficiency of Cks1 in TGF-beta-treated cells likely contributes to the stabilization of p27Kip1 and destabilization of Skp2, because in the absence of Cks1, SCFSkp2 cannot ubiquitinate p27Kip1; instead, self-ubiquitination of Skp2 occurs. Thus, stabilization of the cell cycle inhibitor p27Kip1 and cell growth inhibition in response to TGF-beta occur in part through limiting the threshold of the SCFSkp2 ubiquitin ligase by transcriptional and post-transcriptional mechanisms.

Interleukin-3 in Cooperation with Transforming Growth Factor β Induces Granulocyte Macrophage Colony Stimulating Factor Independent Differentiation of Human CD34+ Hematopoietic Progenitor Cells into Dendritic Cells with Features of Langerhans Cells

Recently, we have reported that M-CSF in cooperation with TGF-beta1 can induce Langerhans cell (LC) development from hematopoietic progenitor cells (HPCs) without GM-CSF. In the present study, we examined whether TGF-beta1 changes the differentiation of HPCs induced by IL-3 towards LC development. We cultured HPCs in a serum-free medium in the presence of IL-3 and a combination cytokines including Flt3L, SCF, and TNF-alpha with or without TGF-beta1. DCs induced by the IL-3 culture (IL-3 DCs) did not significantly differ from those induced by the GM-CSF culture (GM-CSF DCs). Namely, both expressed CDla, F-cadherin, and Langerin in the presence of TGF-beta1 and stimulated allogeneic T cells at a similar magnitude. In contrast to GM-CSF DCs, IL-3 DCs lacked the expression of Birbeck granules (BGs) in spite of their expression of Langerin. When we compared the expression of Langerin between these two DCs, however, it became clear that both Langerin protein and mRNA were significantly lower in IL-3 DCs than in GM-CSF DCs. These studies again demonstrated the ability of TGF-beta1 to polarize the differentiation of HPCs induced by IL-3 towards LC development, although IL-3 DCs were unable to form BGs partly because of their poor ability to induce Langerin.