Absence Of TGF-β Research Articles

TGF beta 1 inhibits Ca2+-calcineurin-mediated activation in thymocytes.

TGFbeta1 is a polypeptide growth modulatory and differentiation factor involved in many biological processes including immune homeostasis and self-tolerance. Tgfb1 knockout mice die around weaning age due to severe inflammation in most major organ systems, but the mechanism underlying this disease is not understood. In this study we demonstrate that Tgfb1(-/-) CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes are hyperresponsive to receptor-mediated and receptor-independent mitogenic stimulation. A suboptimal concentration of ionomycin in the presence of PMA fully activates Tgfb1(-/-) thymocytes, whereas the inhibitors of Ca(2+) influx and calcineurin, EGTA and FK506, eliminate the hyperresponsiveness. Hence, the hypersensitivity of Tgfb1(-/-) thymocytes is due to a lowered threshold for Ca(2+)-dependent activation. Further, we demonstrate that the hypersensitivity of thymocytes results from the absence of TGFbeta1 and not from the inflammatory environment because the thymocytes are hyperresponsive in preinflammatory-stage Tgfb1(-/-) mice. Our results suggest for the first time that TGFbeta1 functions to inhibit aberrant T cell expansion by maintaining intracellular calcium concentration levels low enough to prevent a mitogenic response by Ca(2+)-independent stimulatory pathways alone. Consequently, TGFbeta1 prevents autoimmune disease through a Ca(2+) regulatory pathway that maintains the activation threshold above that inducible by self-MHC-TCR interactions.

Role of high mobility group protein-1 (HMG1) in amyloid-β homeostasis

In Alzheimer’s disease (AD), fibrillar amyloid-β (Aβ) peptides form senile plaques associated with activated microglia. Recent studies have indicated that microglial Aβ clearance is facilitated by several activators such as transforming growth factor-β1 (TGF-β1). The relationship between microglia and Aβ formation and deposition is still unclear. In the present study, high mobility group protein-1 (HMG1) inhibited the microglial uptake of Aβ (1–42) in the presence and absence of TGF-β1. In addition, HMG1 bound to Aβ (1–42) and stabilized the oligomerization. In AD brains, protein levels of HMG1 were significantly increased in both the cytosolic and particulate fractions, and HMG1 and Aβ were colocalized in senile plaques associated with microglia. These results suggest that HMG1 may regulate the homeostasis of extracellular Aβ (1–42) and Aβ oligomerization.

Localization of insulin-like growth factor binding protein-2 in chondrocytes of bovine articular cartilage

Purpose: Previous work indicated that transforming growth factor (TGF-β) treatment of bovine articular cartilage resulted in an accumulation of insulin-like growth factor binding protein-2 (IGF-BP-2). The purpose of the work presented in this paper was to define the localization of the IGF-BP-2 in freshly excised articular cartilage and in slices cultured in the presence and absence of TGF-β. Method: Newborn calf articular cartilage was dissected and immediately fixed or maintained in organ culture for five days under basal conditions (media without added serum or growth factors) or with basal media containing 15 ng/ml of TGF-β1. Frozen or paraffin embedded sections were prepared, and immunohistochemistry using anti-IGF-BP-2 performed. Results: The paraffin sections provided the best preservation of morphology and consistency of immunohistochemical staining patterns. In fresh cartilage slices, IGF-BP-2 was associated with most of the chondrocytes. The basal cultured cartilage showed positive immunostaining in some areas, but not others: the most consistently stained area was the upper radial zone. In all cases where a positive reaction was observed, it was associated mostly with chondrocytes. On the other hand, all the TGF-β treated samples that were examined in this study were evenly stained, and most chondrocytes were positive in all areas from superficial to deep zones, thus closely resembling the pattern of fresh tissue. Conclusions: It is concluded that IGF-BP-2 is closely cell associated in bovine articular cartilage. Following culture of cartilage slices, TGF-β increases the number of cells with positive immunostaining. These data help to support the postulate that TGF-β exerts at least some of its actions in articular cartilage via cross-talk mechanisms involving the IGF-BP-2 system.

TGFbeta induces GDNF responsiveness in neurons by recruitment of GFRalpha1 to the plasma membrane.

We have previously shown that the neurotrophic effect of glial cell line–derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)β. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFβ promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFβ, pretreatment with TGFβ confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFβ-induced recruitment of the glycosyl-phosphatidylinositol-anchored GDNF receptor (GFR)α1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRα1 can promote survival in the absence of TGFβ. Our data suggest that TGFβ is involved in GFRα1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects.

CTGF expression in mesangial cells: Involvement of SMADs, MAP kinase, and PKC

CTGF expression in mesangial cells: Involvement of SMADs, MAP kinase, and PKC.BackgroundThe induction of excess matrix in renal fibrosis seems to be mediated, at least in part, by the transforming growth factor-β (TGF-β)-mediated induction of connective tissue growth factor (CTGF) in mesangial cells.MethodsBy examining CTGF protein and mRNA expression and promoter activity in the presence or absence of TGF-β or inhibitors, the signaling pathways controlling basal and TGF-β–induced CTGF expression in mesangial cells were investigated.ResultsTGF-β enhances CTGF mRNA and protein expression in mesangial cells. Mutation of a consensus SMAD binding element in the CTGF promoter completely abolished TGF-β–induced CTGF expression and reduced basal CTGF expression. The previously identified basal control element-1 (BCE-1) site, but not Sp1 contributes to basal CTGF promoter activity. Ras/MEK/ERK, protein kinase C (PKC) and tyrosine kinase activity also contribute to basal and TGF-β–induced CTGF promoter activity in cultured mesangial cells.ConclusionsThe TGF-β–induction of CTGF in mesangial cells requires SMADs and PKC/ras/MEK/ERK pathways. SMADs are involved in basal CTGF expression, which presumably reflects the fact that mesangial cells express TGF-β endogenously. TGF-β also induces CTGF through ras/MEK/ERK. Inhibiting ras/MEK/ERK seems not to reduce phosphorylation (that is, activation) of SMADs, suggesting that SMADs, although necessary, are insufficient for the TGF-β–stimulation of the CTGF promoter through ras/MEK/ERK. Thus, maximal TGF-β induction of CTGF requires synergy between SMAD and ras/MEK/ERK signaling. CTGF expression in mesangial cells: Involvement of SMADs, MAP kinase, and PKC. The induction of excess matrix in renal fibrosis seems to be mediated, at least in part, by the transforming growth factor-β (TGF-β)-mediated induction of connective tissue growth factor (CTGF) in mesangial cells. By examining CTGF protein and mRNA expression and promoter activity in the presence or absence of TGF-β or inhibitors, the signaling pathways controlling basal and TGF-β–induced CTGF expression in mesangial cells were investigated. TGF-β enhances CTGF mRNA and protein expression in mesangial cells. Mutation of a consensus SMAD binding element in the CTGF promoter completely abolished TGF-β–induced CTGF expression and reduced basal CTGF expression. The previously identified basal control element-1 (BCE-1) site, but not Sp1 contributes to basal CTGF promoter activity. Ras/MEK/ERK, protein kinase C (PKC) and tyrosine kinase activity also contribute to basal and TGF-β–induced CTGF promoter activity in cultured mesangial cells. The TGF-β–induction of CTGF in mesangial cells requires SMADs and PKC/ras/MEK/ERK pathways. SMADs are involved in basal CTGF expression, which presumably reflects the fact that mesangial cells express TGF-β endogenously. TGF-β also induces CTGF through ras/MEK/ERK. Inhibiting ras/MEK/ERK seems not to reduce phosphorylation (that is, activation) of SMADs, suggesting that SMADs, although necessary, are insufficient for the TGF-β–stimulation of the CTGF promoter through ras/MEK/ERK. Thus, maximal TGF-β induction of CTGF requires synergy between SMAD and ras/MEK/ERK signaling.

Vascular Smooth Muscle α-Actin Gene Transcription during Myofibroblast Differentiation Requires Sp1/3 Protein Binding Proximal to the MCAT Enhancer

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor beta1 (TGF-beta1) and accompanied by transient activation of the vascular smooth muscle alpha-actin (SmalphaA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmalphaA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-beta1. However, TGF-beta1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmalphaA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmalphaA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmalphaA enhancer required for TGF-beta1 amplification. Additionally, the TGF-beta1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmalphaA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmalphaA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmalphaA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.

Restoration of TGF-β regulation of plasminogen activator inhibitor-1 in Smad3-restituted human choriocarcinoma cells

Proliferation, migration, and invasiveness of the normal placental extravillous trophoblast (EVT) cells are negatively regulated by transforming growth factor-β (TGF-β), whereas malignant EVT (JAR and JEG-3 choriocarcinoma) cells are resistant to TGF-β. These malignant cells were found to have lost the expression of Smad3. Present study examined whether Smad3 restitution in JAR cells could restore TGF-β response. We produced a stable Smad3 cDNA-transfected clone (JAR-smad3/c) which exhibited further upregulation of Smad3 in the presence of TGF-β1. Since anti-invasive effects of TGF-β in the normal EVT cells were shown to be mediated in part by plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA), we compared the expression of PAI-1 and uPA in the normal EVT, JAR, and JAR-smad3/c cells in the presence or absence of TGF-β1. The basal levels of PAI-1 mRNA and secreted PAI-1 and uPA proteins were found to be very low in JAR and JAR-smad3/c cells, as compared to the normal EVT cells. However, TGF-β1 upregulated PAI-1 and downregulated uPA in JAR-smad3/c cells, but not in JAR cells. Thus, resistance of choriocarcinoma cells to anti-invasive effects of TGF-β may, at least in part, be due to loss of Smad3 expression.

Antagonism of glucocorticoid receptor transactivity and cell growth inhibition by transforming growth factor-β through AP-1-mediated transcriptional repression

We have examined the interaction of the glucocorticoid receptor (GR) and transforming growth factor-β (TGF-β) signal pathways because of their mutual involvement in the regulation of cell growth, development and differentiation. Most studies of this cross-talk event have focused on the effects of glucocorticoids (GCs) on TGF-β responses. In this work, we show that TGF-β can antagonize dexamethasone (Dex)-mediated growth suppression in mouse fibrosarcoma L929 cells. TGF-β also repressed GR-mediated reporter (pMMTV-CAT) gene expression in a concentration-dependent manner, with an IC 50 of 5 ng/ml of TGF-β. Maximal inhibition (76%) was observed at 10 ng/ml of TGF-β. Conversely, Dex inhibited TGF-β-mediated promoter (p3TP-Lux) activity in these same cells. As TGF-β inhibition of GR-mediated gene expression occurred after Dex-mediated nuclear translocation of GR, we conclude that TGF-β inhibition of GR signaling occurs at the level of GR-mediated transcription activity. However, TGF-β did not repress GR-mediated gene expression using the pGRE 2E1B-CAT minimal promoter construct, suggesting that TGF-β did not inhibit intrinsic GR activity but, rather, required DNA-binding factor(s) distinct from GR. As the MMTV promoter contains several putative AP-1 binding sites, we hypothesized that AP-1, a transcription factor composed of c-jun and c-fos proteins, might be involved in the TGF-β inhibition of GR functions. Curcumin, a potent inhibitor of AP-1 expression, completely abolished the inhibitory effect of TGF-β on GR-mediated gene expression without affecting GR activity in the absence of TGF-β, and this drug blocked TGF-β-induced binding of AP-1 to a response element derived from the MMTV sequence. Furthermore, curcumin abolished TGF-β inhibition of Dex-induced growth suppression. Taken as a whole, our data suggest that TGF-β can antagonize the growth inhibitory properties of GR by blocking GR transactivity at complex promoters through a mechanism involving transcriptional repression by DNA-bound AP-1.

The hypertrophic effect of transforming growth factor-beta is reduced in the absence of cyclin-dependent kinase-inhibitors p21 and p27.

Transforming growth factor-beta (TGF-beta) has both antiproliferative and hypertrophic effects on mesangial cells (MC). However, it is not known if these processes are independent or if they share common signaling pathways. Proliferation and hypertrophy are regulated by specific cell-cycle regulatory proteins, where the cyclin-dependent kinase (CDK) inhibitors inhibit target cyclin-CDK complexes. This study examined whether the growth regulatory effects of TGF-beta were determined by the CDK inhibitors p21 and p27. Accordingly, cultured MC from wild type (+/+) and single and double null (-/-) p21 and p27 mice were grown in 5% serum in the presence or absence of TGF-beta1 (2 ng/ml). Proliferation ([(3)H]-thymidine incorporation, cell number, cell cycle) and hypertrophy ([(3)H]-leucine incorporation, total protein content, forward light scatter) were measured after 24 h, 48 h, and 96 h. TGF-beta inhibited proliferation in +/+ and p21/p27 double -/- MC to a similar extent. TGF-beta induced hypertrophy in +/+ MC (18.0% increase at 48 h), and to lesser extent in p21 -/- (12.8%) and p27 -/- MC (11.5%) measured by forward light scatter analysis. In p21/p27 double -/-, the hypertrophic effects of TGF-beta were significantly reduced (3.9% at 48 h). Similar results were obtained by measuring hypertrophy by total protein and [(3)H]-leucine incorporation. In conclusion, the CDK inhibitors p21 and p27 are not required for the antiproliferative effects of TGF-beta. However, the hypertrophic growth effects of TGF-beta are reduced in the absence of both p21 and p27. These data suggest that the regulation of the antiproliferative and hypertrophic effects of TGF-beta may be distinct processes.

DEC1 is a downstream target of TGF-beta with sequence-specific transcriptional repressor activities.

To identify genes that mediate transforming growth factor-beta (TGF-beta) signaling, a colorectal cancer cell line that was sensitive to the growth inhibitory effects of this cytokine was created. We then determined the global gene expression profiles of these cells, and those of HaCaT human keratinocytes, in the presence and absence of TGF-beta. Of the several genes identified in this screen, DEC1 was of particular note in light of the rapidity and consistency of its induction and its potential biochemical activities. We identified a consensus DNA-binding site for DEC1 and showed that DEC1 could repress the transcription of a reporter containing this binding site in its promoter. Finally, both alleles of the DEC1 locus in HaCaT cells were inactivated through targeted homologous recombination. This approach revealed that DEC1 induction was not required for the growth inhibition mediated by TGF-beta in this line. However, DEC1 may function in concert with other signaling components to mediate certain biologic effects of TGF-beta.

Synergistic induction of apoptosis in primary CD4(+) T cells by macrophage-tropic HIV-1 and TGF-beta1.

Depletion of CD4(+) T lymphocytes is a central immunological characteristic of HIV-1 infection. Although the mechanism of such CD4(+) cell loss following macrophage-tropic (R5) HIV-1 infection remains unclear, interactions between viral and host cell factors are thought to play an important role in the pathogenesis of HIV-1 disease. Based on the observation that TGF-beta1 enhanced expression of HIV chemokine coreceptors, the role of this host factor in virus effects was investigated using PBLs cultured in a nonmitogen-added system in the absence or presence of TGF-beta1. Most CD4 cells in such cultures had the phenotype CD25(-)CD69(-)DR(-)Ki67(-) and were CD45RO(bright)CD45RA(dim). Cultured cells had increased expression of CCR5 and CXCR4 and supported both HIV-1 entry and completion of viral reverse transcription. Virus production by cells cultured in the presence of IL-2 was inhibited by TGF-beta1, and this inhibition was accompanied by a loss of T cells from the culture and an increase in CD4(+) T cell apoptosis. Whereas R5X4 and X4 HIV-1 infection was sufficient to induce T cell apoptosis, R5 HIV-1 failed to induce apoptosis of PBLs in the absence of TGF-beta1 despite the fact that R5 HIV-1 depletes CD4(+) T cells in vivo. Increased apoptosis with HIV and TGF-beta1 was associated with reduced levels of Bcl-2 and increased expression of apoptosis-inducing factor, caspase-3, and cleavage of BID, c-IAP-1, and X-linked inhibitor of apoptosis. These results show that TGF-beta1 promotes depletion of CD4(+) T cells after R5 HIV-1 infection by inducing apoptosis and suggest that TGF-beta1 might contribute to the pathogenesis of HIV-1 infection in vivo.

Transforming growth factor β enhances the expression of CD154 (CD40L) and production of tumor necrosis factor α by human T lymphocytes

Activation of lymphocytes in the presence of transforming growth factor beta (TGFβ) can impair or enhance their functional activity. We have found that TGFβ is important in the generation of lymphocytes, which are capable of suppressing antibody production. To better understand how this cytokine affects lymphocyte activity, we looked at the expression of early activation events of T cells stimulated in the presence or absence of TGFβ. The results show that TGFβ enhances the expression of CD154 (CD40L), TNFR2 and the production of TNFα. These findings clarify the co-stimulatory effects of TGFβ that enhance T lymphocyte activation.

Transforming Growth Factor-β1 Inhibits Vascular Permeability Factor Release by T Cells in Normal Subjects and in Patients with Minimal-Change Nephrotic Syndrome

Background/Aim: A lymphokine, the vascular permeability factor (VPF), which increases vascular permeability, has been characterized in minimal-change nephrotic syndrome (MCNS). Transforming growth factor-β (TGF-β) is an immunosuppressive cytokine that inhibits proliferation, cytokine production, and cytotoxic activity of T cells and natural killer cells. We, therefore, investigated the effects of TGF-β₁ on the release of VPF by peripheral blood T cells from MCNS patients. The aim of our study was to determine the in vitro immunosuppressive capacity of TGF-β₁ in patients with MCNS. Methods: To further test the effect of TGF-β₁ on concanavalin A (Con A)-induced VPF release, normal and MCNS T cells were stimulated with 5 µg/ml of Con A in the presence or absence of TGF-β₁, and the culture supernatants were tested for the presence of VPF by the method of Lagrue et al. The disease controls included 16 patients with IgA nephropathy. Results: Significantly increased spontaneous and Con A-stimulated secretion of VPF was detected in T-cell cultures of MCNS patients with the nephrotic syndrome as compared with those of normal controls. Addition of TGF-β₁ to these cultures inhibited the release of VPF in a dose-dependent manner. The effect of TGF-β₁ on the release of VPF was specific, since a reversion was obtained with a neutralizing monoclonal antibody to human TGF-β₁. Conclusion: Together, our data demonstrate that TGF-β₁ antagonizes the ability of T cells to release VPF, and suggest a role of TGF-β₁ in the pathophysiology of VPF in vitro.

Inhibition of the Transforming Growth Factor β1 Signaling Pathway by the AML1/ETO Leukemia-associated Fusion Protein

The t(8;21) translocation, found in adult acute myelogenous leukemia, results in the formation of an AML1/ETO chimeric transcription factor. AML1/ETO expression leads to alterations in hematopoietic progenitor cell differentiation, although its role in leukemic transformation is not clear. The N-terminal portion of AML1, which is retained in AML1/ETO, contains a region of homology to the FAST proteins, which cooperate with Smads to regulate transforming growth factor beta1 (TGF-beta1) target genes. We have demonstrated the physical association of Smad proteins with AML1 and AML1/ETO by immunoprecipitation and have mapped the region of interaction to the runt homology domain in these AML1 proteins. Using confocal microscopy, we demonstrated that AML1, and ETO and/or AML1/ETO, colocalize with Smads in the nucleus of t(8;21)-positive Kasumi-1 cells, in the presence but not the absence of TGF-beta1. Using transient transfection assays and a reporter gene construct that contains both Smad and AML1 consensus binding sequences, we demonstrated that overexpression of AML1B cooperates with TGF-beta1 in stimulating reporter gene activity, whereas AML1/ETO represses basal promoter activity and blocks the response to TGF-beta1. Considering the critical role of TGF-beta1 in the growth and differentiation of hematopoietic cells, interference with TGF-beta1 signaling by AML1/ETO may contribute to leukemogenesis.

Novel Cdk Inhibitors Restore TGF-β Sensitivity in Cdk4 Overexpressing Epithelial Cells

Transforming growth factor-beta (TGF-β) is a potent mitogen that effects a wide variety of cells by blocking cell growth. TGF-β acts by interacting with components of cell cycle machinery to cause G1 arrest and in mink lung epithelial cells (Mv1Lu) it does so by inhibiting Cdk4 synthesis. Overexpression of Cdk4 in these cells (B7) renders them resistant to the effects of TGF-β. Here we report that two novel Cdk inhibitors (pyridopyrimidines) that not only inhibit Cdk4 and Cdk2 in an in vitro kinase assay but also, in the absence of TGF-β, block growth of Mv1Lu cells in G1 more efficiently than their B7 (overexpressing Cdk4) counterparts. Interestingly, these inhibitors restored sensitivity of B7 cells towards TGF-β. This may have implications for the treatment of tumors that have lost TGF-β responsiveness due to deregulated cellular growth in vivo. These Cdk inhibitors could therefore be used in conjunction with TGF-β to understand the mechanism of growth arrest in normal versus tumour cells.

Inhibition of type I and III IP3Rs by TGF-β is associated with impaired calcium release in mesangial cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) mediate cytosolic free calcium concentration ([Ca(2+)](c)) signals in response to a variety of agonists that stimulate mesangial cell contraction and proliferation. In the present study, we demonstrate that mesangial cells express both type I and III IP(3)Rs and that these receptors occupy different cellular locations. Chronic treatment with transforming growth factor-beta1 (TGF-beta1; 10 ng/ml, 24 h) leads to downregulation of both type I and III IP(3)Rs as measured by immunoblot and confocal analysis. TGF-beta1 treatment does not affect IP(3) levels, and downregulation of type I IP(3)R is not due to enhanced degradation of the protein, as the half-life of type I IP(3)R is unchanged in the presence or absence of TGF-beta1. Functional effects of TGF-beta1-induced downregulation of the IP(3)Rs were evaluated by measuring [Ca(2+)](c) changes in response to epidermal growth factor (EGF) in intact cells and sensitivity of [Ca(2+)](c) release to IP(3) in permeabilized cells. TGF-beta1 pretreatment led to a significant decrease of [Ca(2+)](c) release induced by EGF in intact cells and by submaximal IP(3) (400 nm) in permeabilized cells. Total IP(3)-sensitive [Ca(2+)](c) stores were not changed, as assessed by stimulation with maximal doses of IP(3) (10.5 microm) and thapsigargin-mediated calcium release in permeabilized cells. We conclude that prolonged exposure to TGF-beta1 leads to downregulation of both type I and III IP(3)Rs in mesangial cells and this is associated with impaired sensitivity to IP(3).

Selectively increased growth of fetal hemoglobin-expressing adult erythroid progenitors after brief treatment of early progenitors with transforming growth factor beta

We have studied the effect of transforming growth factor beta (TGFbeta) on erythropoiesis in cultures from adult peripheral blood, using flow cytometric enumeration of fetal hemoglobin (HbF)-containing cells. TGFbeta caused a dramatic increase in the proportions of cells that accumulated HbF together with adult hemoglobin (HbA) (F+A+ cells). This highly significant (P <.0001) increase in F+ cell proportion was achieved by TGFbeta treatment during the first 4 days of culture and was sustained during further culture expansion in the absence of TGFbeta. The increase in F+ cell proportions did not depend on the cytokine combination (EPO+SCF+IL3, EPO+SCF, EPO+IL3, SCF+IL3) used during the phase of TGFbeta treatment. Increased F+ cell proportions were paralleled by an increased molecular ratio of HbF/ HbF+ HbA, measured by cation exchange high-performance liquid chromatography (HPLC). In addition to the effect on F+ cell proportions, TGFbeta caused a dramatic increase in overall cell division potential. By the time cultures reached terminal growth arrest (12-14 days in controls and 18-26 days after TGFbeta), the overall numbers of F+ cells produced per initially seeded clonogenic cell was approximately 10 times higher in the TGFbeta-treated cultures than in the controls. We propose to investigate whether the TGFbeta-induced increase in relative and absolute numbers of nucleated F+ cells, as demonstrated in vitro, can be translated into increased F+ erythrocytes in vivo, allowing therapeutic application for some beta-hemoglobinopathies. (Blood. 2000;95:2967-2974)

Respective involvement of TGF-beta and IL-4 in the development of Langerhans cells and non-Langerhans dendritic cells from CD34+ progenitors.

In vivo, dendritic cells (DC) form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells dependent on transforming growth factor beta(TGF-beta) for its development. In this study, we show that endogenous TGF-beta is required for the development of both LC and non-LC DC from CD34+ hematopoietic progenitor cells (HPC) through induction of DC progenitor proliferation and of CD1a+ and CD14+ DC precursor differentiation. We further demonstrate that addition of exogenous TGF-beta polarized the differentiation of CD34+ HPC toward LC through induction of differentiation of CD14+ DC precursors into E-cadherin+, Lag+CD68-, and Factor XIIIa-LC, displaying typical Birbeck granules. LC generated from CD34+ HPC in the presence of exogenous TGF-beta displayed overlapping functions with CD1a+ precursor-derived DC. In particular, unlike CD14(+)-derived DC obtained in the absence of TGF-beta, they neither secreted interleukin-10 (IL-10) on CD40 triggering nor stimulated the differentiation of CD40-activated naive B cells. Finally, IL-4, when combined with granulocyte-macrophage colony-stimulating factor (GM-CSF), induced TGF-beta-independent development of non-LC DC from CD34+ HPC. Similarly, the development of DC from monocytes with GM-CSF and IL-4 was TGF-beta independent. Collectively these results show that TGF-beta polarized CD34+ HPC differentiation toward LC, whereas IL-4 induced non-LC DC development independently of TGF-beta.

Cell proliferation: Interfering with Smads

Unregulated cell growth results in tumor production; therefore, it is important to identify developmental factors that regulate cell growth. Transforming growth factor-beta (TGF-β) regulates cell growth and differentiation through receptor-mediated phosphorylation of Smad proteins. These Smad proteins in turn form protein complexes and enter the cell's nucleus to activate the transcription of target genes. Stroschein et al. have identified a new player, SnoN, in TGF-β signaling. In the absence of TGF-β, the SnoN oncoprotein binds to a Smad2/Smad4 complex and recruits a transcription co-repressor, thus inhibiting transcription activation. When TGF-β is present, Smad3 triggers the degradation of SnoN and transcription activation resumes. Finally, a negative-feedback mechanism is present in which TGF-β stimulates SnoN production, which then represses the transcription activation function of the Smad complex once again. SnoN is found in various carcinomas. Hence, the transforming activity of SnoN may be explained by its interference with the role of TGF-β in inhibiting cell growth.Stroschein, S.L., Wang, W., Zhou, S., Zhou, Q., and Luo, K. (1999) Negative feedback regulation of TGF-signaling by the SnoN oncoprotein. Science 286: 771 - 774. [Abstract] [Full Text]

Cell proliferation: Interfering with Smads

Unregulated cell growth results in tumor production; therefore, it is important to identify developmental factors that regulate cell growth. Transforming growth factor-beta (TGF-β) regulates cell growth and differentiation through receptor-mediated phosphorylation of Smad proteins. These Smad proteins in turn form protein complexes and enter the cell's nucleus to activate the transcription of target genes. Stroschein et al . have identified a new player, SnoN, in TGF-β signaling. In the absence of TGF-β, the SnoN oncoprotein binds to a Smad2/Smad4 complex and recruits a transcription co-repressor, thus inhibiting transcription activation. When TGF-β is present, Smad3 triggers the degradation of SnoN and transcription activation resumes. Finally, a negative-feedback mechanism is present in which TGF-β stimulates SnoN production, which then represses the transcription activation function of the Smad complex once again. SnoN is found in various carcinomas. Hence, the transforming activity of SnoN may be explained by its interference with the role of TGF-β in inhibiting cell growth. Stroschein, S.L., Wang, W., Zhou, S., Zhou, Q., and Luo, K. (1999) Negative feedback regulation of TGF-signaling by the SnoN oncoprotein. Science 286 : 771 - 774. [Abstract] [Full Text]