Neutralization Of TGF-beta Research Articles

Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function.

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.

Endothelial–Mesenchymal Interactions In Vitro Reveal Molecular Mechanisms of Smooth Muscle/Pericyte Differentiation

Cell-cell interactions are central to vascular development. We have developed an in vitro system in which endothelial cells (EC) are co-cultured with 10T1/2 cells as smooth muscle cell (SMC)/pericyte precursors. 10T1/2 cells, in contact with EC, differentiate to SMC in a process mediated, at least in part, by a transforming growth factor-beta (TGF-beta)-mediated event. Co-culture with EC or TGF-beta treatment induced expression of SM22alpha, with co-culture inducing a significantly greater response. To dissect the molecular mechanisms of SMC/pericyte differentiation, reporter constructs containing the promoter for SM22alpha, a SMC-specific gene, were stably transfected into 10T1/2 cells and response to EC-co-culture and TGFbeta were compared. Co-culture with EC or TGFbeta treatment stimulated activity of a 441-bp SM22-alpha promoter to about the same extent, whereas co-culture induced the activity of a 3.7-kb promoter to about twice that of TGBbeta. Neutralization of TGFbeta in EC-10T1/2 co-cultures partially reduced the 3.7-kb SM22alpha promoter activity in 10T1/2 cells. Previously unidentified CArG and TCE elements near the 5' end of the promoter are responsible for full promoter activity. EC-mesenchymal contact appears to be required for full promoter activity of the SM22alpha gene in 10T1/2 and requires upstream CArG and TCE elements. The 3.7-kb SM22alpha promoter can direct expression of lacZ in vivo to SMC of the large vessels and the smaller intersomitic vessels. We have identified the expression of SM22alpha in pericytes of the retinal microvasculature in developing and remodeling vessels.

Localization and early time course of TGF‐β1 mRNA expression in dystrophic muscle

Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy (DMD). In the dystrophic (mdx) mouse model of DMD, the diaphragm is more severely affected than other skeletal muscles. The level of transforming growth factor-beta1 (TGF-beta1), an inflammatory cytokine, is significantly elevated in mdx diaphragm. However, little is known about the onset of TGF-beta1 messenger ribonucleic acid (mRNA) expression, or which cells express the mRNA. In this study, we characterized the location and time course of expression of TGF-beta1 mRNA in diaphragm from mdx mice. TGF-beta1 mRNA was significantly elevated in mdx diaphragm at 6 and 9 but not 12 weeks of age, and these changes corresponded with changes in type I collagen mRNA and hydroxyproline concentration. Mononucleated cells localized to areas of fiber necrosis highly expressed the TGF-beta1 transcript in mdx diaphragm. Neutralization of TGF-beta1 by decorin administration resulted in a 40% reduction in the level of diaphragm muscle type I collagen mRNA. These findings support a role for TGF-beta1 during the early stages of fibrogenesis in dystrophic diaphragm muscle. Therapeutic interventions aimed at neutralizing this cytokine may be beneficial in slowing the development of fibrosis in DMD.

The effects of transforming growth factor-beta neutralization on postburn humoral immunity.

Burn injury induces immunosuppression, which is associated with an increased susceptibility to infection. Our laboratory has demonstrated that burn injury also impairs humoral immunity. We reported that burn injury enhanced expression of transforming growth factor-beta (TGF-beta) mRNA and that exogenous TGF-beta further impaired humoral immunity. The objective of this study was to clarify the role of TGF-beta on humoral immunity after burn injury with a neutralizing experiment. Twelve BALB/c mice were randomly divided into two groups: sham and burn. Anesthetized mice received a 20% full-thickness burn or sham injury. The murine splenocytes containing 1.5 x 10 cells/mL were cultured with 2.5 microg/mL of lipopolysaccharide with or without 0.5 ng/mL of TGF-beta or 1 microg/mL of anti-TGF-beta neutralizing antibody, if necessary. Concentrations of immunoglobulin (Ig) M in the cell culture supernatant were determined by enzyme-linked immunosorbent assay and the number of IgM-secreting cells in the culture was measured by enzyme-linked immunospot assay. After 2-day culture, neutralization of TGF-beta dramatically restored IgM synthesis after burn injury. After 5-day culture, however, it restored IgM concentration but failed to restore a number of IgM-secreting cells. This neutralizing experiment demonstrated that TGF-beta is one of the inhibitors of IgM synthesis after burn injury. However, neutralization of TGF-beta was not enough to completely restore humoral immunity after burn injury. Investigation of the mechanism of impaired IgM synthesis after burn injury should be continued.

TGF-beta signaling in pancreatic cell differentiation

Abstract Introduction: TGF-beta signaling has been implicated in pancreatic cancer due to the prevalence of smad4 mutations. An established model for pancreatic endocrine differentiation is exendin-4 (glucagon-like peptide-1 analog) treatment of AR42J cells (rat pancreatic carcinoma cell line). We studied TGF-beta superfamily signaling and downstream intracellular effectors (smad proteins) implicated in many developmental processes. Here, we found a novel interdependence between GLP-1 and TGF-beta signaling essential for endocrine differentiation. Methods: AR42J cells were cultured with exendin-4 at serial doses for three days. For inhibitory studies, morpholino antisense and missense controls were added to culture media. Other bioactivity studies were performed using either TGF-beta ligands or else a pan-specific TGF-beta neutralizing antibody. Cell differentiation was analyzed by semi-quantitative PCR, as well as immunocytochemistry (IFCC). Results: Smad2 and smad3 mRNA levels were respectively decreased and increased in response to exendin-4 treatment. Further, smad2 antisense or smad2/smad3 double antisense prevented AR42J cells from becoming insulin-positive. Surprisingly, smad3 antisense alone led to a 6.5-fold enhancement of insulin mRNA levels and a doubling of insulin-positive cells by IFCC. The addition of TGF-beta1 ligand greatly upregulated smad3 mRNA and led to a dramatic elevation in insulin expression. Interestingly, TGF-beta smads and insulin-positive differentiation were completely blocked by TGF-beta antibodies, or TGF-beta receptor type I antisense (Alk-1 and Alk-5). Conclusions: TGF-Beta isoform signaling represents a key control point in regulating the level of differentiation of pancreatic AR42J cancer cells.

Shockwave stimulates oxygen radical-mediated osteogenesis of the mesenchymal cells from human umbilical cord blood.

Human umbilical cord blood (HUCB) mesenchymal progenitor cells expressed stro-1 or CD44 or CD29, and subsequently, differentiated toward osteogenic lineage. Physical shockwave treatment increased osteogenic activity of HUCB mesenchymal progenitor cells through superoxide-mediated TGF-beta1 induction. Transplantation of shockwave-treated HUCB mesenchymal progenitor cells enhanced healing of segmental femoral defect in severe combined immunodeficiency disease (SCID) mice. Mesenchymal progenitor cells (MPCs) in the bone marrow are precursors to bone development. It remains uncertain whether MPCs are present in human umbilical cord blood (HUCB) and are capable of differentiating into osteogenic cell lineage. Extending from a model of shockwave (SW) promotion of bone marrow stromal cell differentiation toward osteoprogenitors in rats, we further investigated how physical SW mediated biological responses in regulating osteogenic differentiation of HUCB MPCs. HUCB was subjected to SW treatment at different energy flux densities and impulses. Colony-forming units-stroma (CFU-Stroma), osteogenic activities (Cbfa1/Runx2 expression, bone alkaline phosphatase activity, and bone nodule formation), and bone formation by heterologous transplantation into SCID mice were assessed. Few CD34+ stem cells (1.3%) and stro-1+ cells (1.0%) were present in the freshly prepared mononuclear cells (MNCs) from HUCB. The number of stro-1+ cells, but not CD34+, increased to 72.4% in the adherent cell culture over 6 days. Stro-1+ cells co-expressed CD44 and CD29 markers and grew into CFU-Stroma that matured into bone nodules. We found that the SW treatment (0.16 mJ/mm2 energy flux density, 200 impulses) elicited superoxide production and promoted formation of CFU-Stroma, but not of hematopoietic CFU-Mix. SW also enhanced the production of transforming growth factor (TGF)-beta1, but not of interleukin (IL)-3 or granulocyte monocyte-colony stimulating factor (GM-CSF). Neutralization of TGF-beta1 significantly reduced SW-promoted CFU-Stroma formation. Superoxide scavenging by superoxide dismutase blocked SW enhancement of TGF-beta1 production and formation of CFU-Stroma. Administration of SW-treated HUCB MPCs to SCID mice with femoral segmental defects facilitated dense, bridging callus and gap closure. HUCB MPCs subjected to SW treatment is a potential source for stem cells useful in the treatment of orthopedic disorders. An optimal physical SW treatment enhanced osteogenesis through superoxide-mediated TGF-beta1 production. Physical stimulation is an alternative method for extending mesenchymal stem cells of HUCB.

The induction of TGF-beta(1) and NF-kappaB parallels a biphasic time course of leukocyte/endothelial cell adhesion following low-dose X-irradiation.

Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the knowledge of the underlying molecular mechanisms is still scarce. The authors have recently reported that transforming growth factor beta 1 (TGF-beta(1)) essentially contributes to a reduced endothelial adhesion of mononuclear cells (PBMC) following LD-RT. Furthermore, TGF-beta(1) secretion was associated with the induction of the transcription factor nuclear factor kappa B (NF-kappaB). However, the time course of adhesion, TGF-beta(1) expression, and NF-kappaB activity following LD-RT has not been thoroughly investigated yet. The human EA.hy.926 endothelial cell line (EA.hy.926 EC) was grown to 95% confluence. Immediately after stimulation with the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), EA.hy.926 EC were irradiated with single doses ranging from 0.3 up to 3 Gy. Adhesion assays were performed 4, 12, and 24 h after irradiation. Nuclear extracts and culture supernatants were harvested after 4, 8, 12, 20, 24, 30, and 40 h. NF-kappaB DNA-binding activity was analyzed by electrophoretic mobility shift assays (EMSA) and TGF-beta(1) secretion by enzyme-linked immunosorbent assay (ELISA). The functional impact of TGF-beta(1) on the course of leukocyte/EC adhesion was analyzed by neutralizing TGF-beta(1) in parallel with stimulation/irradiation of the EA.hy.926 EC. 4 and 24 h after irradiation of EA.hy.926 EC in the dose range between 0.3 and 0.7 Gy, a reduced adhesion of PBMC compared to nontreated controls could be observed. However, 12 h after irradiation a relative maximum of adhesion (up to 30% increase) was seen at a dose of 0.3 Gy. TGF-beta(1) secretion and NF-kappaB DNA-binding activity displayed a similar biphasic kinetics of induction with a relative minimum 12 h after irradiation. Neutralization of TGF-beta(1) activity restored adhesion at 4 and 24 h after LD-RT of EA.hy.926 EC, but it did not influence leukocyte adhesion 12 h after irradiation. LD-RT of stimulated human EA.hy.926 EC is followed by a biphasic time course of NF-kappaB activity and an increased secretion of TGF-beta(1). The kinetics shows peak levels at 4-8 h and 24-30 h after LD-RT and results in a biphasic leukocyte/EC adhesion profile.

Overexpression of latent transforming growth factor-beta binding protein 1 (LTBP-1) in dioxin receptor-null mouse embryo fibroblasts.

The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type (AhR+/+) and null (AhR-/-) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-beta binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR-/- MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1, again suggesting an AhR-regulated mechanism. Treatment of AhR+/+ MEF with transforming growth factor-beta(TGF-beta) downregulated AhR and, simultaneously, increased Ltbp-1, further supporting the role of this receptor in LTBP-1 expression. AhR-/- conditioned medium had higher levels of active and total TGF-beta activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-beta concentrations. TGF-beta did not appear to directly regulate Ltbp-1 given that addition of TGFbeta neutralizing antibody or TGFbeta protein to AhR-/- MEF had no effect on Ltbp-1 expression. AhR-/- MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMP-inhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFbeta activity and cell proliferation.

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

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

Regulation of autoimmune diabetes by non-islet-specific T cells - a role for the glucocorticoid-induced TNF receptor.

Diabetogenic BDC2.5 CD4 T cells induce diabetes when injected into NOD.scid mice. However, when co-transferred with the OVA-specific DO11.10 CD4 T cells, BDC2.5 T cells failed to cause diabetes. This inhibition depended upon the stimulation of DO11.10 T cells only with soluble OVA, which skewed their differentiation to a Th2-type pattern of cytokine secretion in vivo. However, in vivo neutralization of IL-4, IL-10 or TGF-beta using monoclonal antibodies did not prevent the inhibition whereas treatment with an antibody against the glucocorticoid-induced TNF receptor abrogated the protection from disease. In the protected mice, the diabetogenic T cells could be isolated from their spleens and shown to transfer diabetes when injected into new NOD.scid recipients. Thus, the inhibition took place without the physical or functional elimination of the diabetogenic T cells.

Serum derived from multiple trauma patients promotes the differentiation of endothelial progenitor cells in vitro: possible role of transforming growth factor-beta1 and vascular endothelial growth factor165.

Ischemia in various organs and tissues takes place during and as a direct result of multiple trauma (MT). Bone marrow-derived endothelial progenitor cells (EPCs) are involved in neovascularization after ischemic incidences. Here, we report that serum derived from patients with MT stimulates differentiation of EPCs in vitro from peripheral blood mononuclear cells (PBMCs). EPCs were identified by DiL-Acetyl-LDL-uptake with concomitant UEA-I-lectin binding. A significant increase in EPC numbers was noted when PBMCs were cultivated for 72 h with the serum of MT patients (n = 25) obtained at 5 days. Furthermore, serum from MT patients enhanced the functional acting of EPCs to form prevascular structures in matrigel. Reverse transcription polymerase chain reaction analysis revealed gene expression of transforming growth factor (TGF)-beta1- and vascular endothelial growth factor (VEGF) receptors 1 and 2. Reverse transcription polymerase chain reaction analysis was based on further cultivated cell preparations, which contained at least 80% EPCs. Moreover, the addition of recombinant VEGF or low concentrations of TGF-beta increased EPC differentiation. In addition, neutralization of TGF-beta1 and of VEGF165 in MT serum using specific antibodies resulted in a significant decrease in EPC differentiation. Our data indicate that TGF-beta1 and VEGF165 play a pivotal role for EPC differentiation induced by serum of polytrauma patients.

Divergent effects of low versus high dose anti-TGF-β antibody in puromycin aminonucleoside nephropathy in rats

Transforming growth factor-beta (TGF-beta) modulates immune/inflammatory cells, promotes extracellular matrix (ECM) accumulation, and is increased in fibrotic organs. Here we report the effects of administering a puromycin aminonucleoside nephropathy (PAN)-specific TGF-beta neutralizing antibody on glomerulosclerosis in vivo. Adult male Sprague-Dawley rats underwent uninephrectomy (Nx) followed by intraperitoneal PAN at weeks 2, 6, 7 and 8. Rats were treated with either high (5 mg/kg body weight) (N= 9) or low (0.5 mg/kg body weight) (N= 7) dose TGF-beta antibody intraperitoneally three times weekly until sacrifice at week 10. A PAN untreated control group (N= 7) was dosed with an isotype specific, null antibody. The nephrectomy samples were studied as normal kidney control (NL) (N= 5). Rats undergoing left kidney Nx (N= 5) only were also included as age-matched control. Renal function and morphology were assessed, and molecular studies performed. Systolic blood pressure was increased in parallel over time in all groups (at 10 weeks, control 137 +/- 10 mm Hg; high 129 +/- 4 mm Hg; low 137 +/- 3 mm Hg) (P= NS). Both TGF-beta antibody treatments decreased renal cortex mRNA expressions similarly for TGF-beta1, TGF-beta2, and collagen III (TGF-beta1, control 0.36 +/- 0.02 mm Hg; high 0.19 +/- 0.01 mm Hg; low 0.19 +/- 0.02 mm Hg; P < 0.01 low and high vs. control; TGF-beta2, control 0.38 +/- 0.03 mm Hg; high 0.19 +/- 0.02 mm Hg; low 0.20 +/- 0.03 mm Hg; P < 0.01 low and high vs. control; and collagen III, control 0.33 +/- 0.01 mm Hg; high 0.14 +/- 0.01 mm Hg; low 0.19 +/- 0.01 mm Hg; P < 0.01 low and high vs. control; P < 0.05 low vs. high, data expressed as mRNA normalized density units vs. 18S RNA). However, only low dose TGF-beta antibody improved renal function and sclerosis measured by serum creatinine and creatinine clearance (serum creatinine, control 2.3 +/- 0.5 mg/dL; high 2.5 +/- 0.5 mg/dL; low 0.8 +/- 0.1 mg/dL; P < 0.05 low vs. control and high; creatinine clearance, control 0.44 +/- 0.11 mL/min; high 0.70 +/- 0.26 mL/min; low 1.34 +/- 0.30 mL/min; P < 0.05 low vs. control, P= NS vs. high). In parallel, sclerosis index (0 to 4+ scale) was improved in low dose (control 2.67 +/- 0.27; high 2.37 +/- 0.30; low 1.78 +/- 0.24; P < 0.05 low vs. control). This improved function and structure was linked to decreased glomerular infiltrating macrophages (0 to 4+ score, control 2.3 +/- 0.2; high 1.8 +/- 0.4; low 0.8 +/- 0.1; P < 0.01 low vs. control; P < 0.05 low vs. high; P= NS high vs. control). Further, plasminogen activator inhibitor-1 (PAI-1) mRNA expression in renal cortex was attenuated after low dose TGF-beta antibody treatment compared to control and high dose group (PAI-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio, NL 0.18 +/- 0.003; control 0.45 +/- 0.03; high 0.40 +/- 0.04; low 0.23 +/- 0.01; P < 0.05 low vs. control and high). Matrix metalloproteinase-9 (MMP-9) activity was maintained at higher levels in kidneys of the low dose TGF-beta antibody-treated group. These results show an in vivo dose-response with an agent that blocks the biologic activity of TGF-beta. Higher dose of TGF-beta antibody was without beneficial effect, suggesting that TGF-beta-mediated effects on PAI-1 and macrophage influx are bimodal and closely regulated. Given that both antibody doses reduced the expression of TGF-beta isoforms and collagen III production, but only low dose ameliorated histologic sclerosis, it appears that pharmacologic effects of anti-TGF-beta antibody on matrix synthesis and degradation are not equivalent.

Bone marrow stroma damage induced by chemotherapy for acute lymphoblastic leukemia in children.

Several studies have suggested a role of bone marrow stroma injury in long-term chemotherapy-induced hematopoietic failure. To evaluate whether bone marrow microenvironment is altered by chemotherapy for acute lymphoblastic leukemia (ALL) and to determine its contribution to postchemotherapy anemia, we investigated the ability of stroma from children receiving maintenance chemotherapy for ALL to support hematopoiesis. Long-term bone marrow cultures (LTBMC) were established with bone marrow cells either from ALL children under therapy (n = 24) or from control subjects (n = 19). Nonadherent cells and colony forming units-granulocytic monocytic (CFU-GM) output in LTBMC did not differ between patients and controls. In contrast, burst forming unit-erythroid (BFU-E) numbers were lower in patient LTBMC (p = 0.013). Co-cultures of normal CD34+ cells and preformed patient or control stromas showed significantly reduced hematopoietic supportive capabilities of patient stromas: both CFU-GM and BFU-E were reduced (p = 0.002 and 0.046, respectively). In addition, supernatants (SN) of patients' LTBMC inhibited normal BFU-E growth compared with SN of normal LTBMC. Transforming growth factor (TGF)-beta1 levels were increased in patient cultures (p = 0.0039) and inversely correlated with BFU-E produced in LTBMC (r = -0.36, p = 0.04). Neutralization of TGF-beta1 significantly increased the BFU-E output of patient LTBMC (p = 0.0078). In contrast, macrophage inflammatory peptide (MIP)-1alpha levels were lower in SN of patients compared with controls (p = 0.015). Thus, chemotherapy for ALL induces functional deregulation within bone marrow stromal cells with an increase in the growth-inhibiting factor TGF-beta1, together with a decrease in MIP-1alpha, which might contribute to hematopoietic toxicity.

Induction of antiproliferation and apoptosis in estrogen receptor negative MDA-231 human breast cancer cells by mifepristone and 4-hydroxytamoxifen combination therapy: A role for TGFβ1

Mifepristone (MIF) is an antiprogestin with potent anti-glucocorticoid and anti-androgen activity. MIF also appears to have anti-tumor activity independent of its ability to bind to nuclear receptors. In this study, we tested the ability of MIF to inhibit the growth of ER and PR negative breast cancer cells. In addition, because high-dose anti-estrogen treatment has been shown to inhibit ER and PR negative breast cancer cells, we compared the anti-proliferative activity of MIF to that of the anti-estrogen 4-hydroxytamoxifen (TAM) or combination hormonal therapy (MIF + TAM). MIF and TAM therapy induced a significant time- and dose-dependent growth inhibition and, ultimately, induced cell death in MDA-231 cells as evidenced by increased DNA fragmentation, cytochrome c release from the mitochondria, and the activation of caspase-3. The anti-proliferative activity of TAM plus MIF combination treatment was at least additive as compared to either monotherapy. The earliest indicator of TAM and MIF cytostatic and cytotoxic action on MDA-231 cells was a significant (p<0.05) induction of TGFbeta1 secretion into the growth medium within 4 h of treatment. Secreted TGFbeta1 levels at 24 and 48 h were significantly higher in the TAM plus MIF treatment group as compared to cells treated with TAM or MIF alone. TGFbeta1 neutralizing antibody or addition of mannose-6-phosphate (M6P), a reagent also used to inhibit TGFbeta1, significantly attenuated the TAM and/or MIF-induced cell growth inhibition and cell death. In summary, our results indicate that MIF used in combination with TAM can effectively kill estrogen-insensitive human breast cancer cells. Our study further implies that agents that effectively increase TGFbeta1 levels in ER negative breast cancer cells may be one treatment approach for hormone-independent breast cancers.

SPARC inhibits epithelial cell proliferation in part through stimulation of the transforming growth factor-beta-signaling system.

Secreted protein, acidic and rich in cysteine (SPARC) is a multifunctional secreted protein that regulates cell-cell and cell-matrix interactions, leading to alterations in cell adhesion, motility, and proliferation. Although SPARC is expressed in epithelial cells, its ability to regulate epithelial cell growth remains largely unknown. We show herein that SPARC strongly inhibited DNA synthesis in transforming growth factor (TGF)-beta-sensitive Mv1Lu cells, whereas moderately inhibiting that in TGF-beta-insensitive Mv1Lu cells (i.e., R1B cells). Overexpression of dominant-negative Smad3 in Mv1Lu cells, which abrogated growth arrest by TGF-beta, also attenuated growth arrest stimulated by SPARC. Moreover, the extracellular calcium-binding domain of SPARC (i.e., SPARC-EC) was sufficient to inhibit Mv1Lu cell proliferation but not that of R1B cells. Similar to TGF-beta and thrombospondin-1, treatment of Mv1Lu cells with SPARC or SPARC-EC stimulated Smad2 phosphorylation and Smad2/3 nuclear translocation: the latter response to all agonists was abrogated in R1B cells or by pretreatment of Mv1Lu cells with neutralizing TGF-beta antibodies. SPARC also stimulated Smad2 phosphorylation in MB114 endothelial cells but had no effect on bone morphogenetic protein-regulated Smad1 phosphorylation in either Mv1Lu or MB114 cells. Finally, SPARC and SPARC-EC stimulated TGF-beta-responsive reporter gene expression through a TGF-beta receptor- and Smad2/3-dependent pathway in Mv1Lu cells. Collectively, our findings identify a novel mechanism whereby SPARC inhibits epithelial cell proliferation by selectively commandeering the TGF-beta signaling system, doing so through coupling of SPARC-EC to a TGF-beta receptor- and Smad2/3-dependent pathway.

Aldosterone inhibits inducible nitric oxide synthase in neonatal rat cardiomyocytes.

In studies of animals, increases in aldosterone are associated with myocardial necrosis and fibrosis, and treatment with spironolactone, an antagonist of aldosterone, improved clinical outcomes in patients with heart failure. In the present study, we explored nitric oxide (NO), a signaling molecule involved in cardiac function, as a potential mediator of aldosterone's effects on the heart. Levels of both inducible NO synthase (iNOS) and NO from isolated rat neonatal cardiomyocytes pretreated with IL-1 were found to be decreased with exposure to aldosterone or dexamethasone in a dose-dependent manner. Spironolactone increased iNOS expression and prevented inhibition by aldosterone, consistent with a mineralocorticoid receptor-mediated mechanism for iNOS down-regulation. Aldosterone had no effect on iNOS mRNA levels, indicating a posttranscriptional mechanism for the inhibition of iNOS. Neutralization of TGF-beta 1 using a specific antibody reversed aldosterone-dependent iNOS and NO down-regulation. In summary, aldosterone inhibited IL-1-induced iNOS expression posttranscriptionally by a TGF-beta -dependent mechanism. The decrease in NO synthesis could have relevance to known cardiac effects of aldosterone.

Targeted engagement of CTLA-4 prevents autoimmune thyroiditis.

The CTLA-4-mediated signal is a critical step in the down-modulation of immune responses. The therapeutic potential of this signal to induce tissue-specific tolerance was investigated by using an anti-CTLA-4 antibody that was coupled to an antibody specific for the thyrotropin receptor. After in vivo administration, this bispecific antibody (BiAb) accumulated in the thyroid and prevented development of experimental autoimmune thyroiditis (EAT) in mice immunized with mouse thyroglobulin (mTg). Lymphocytes from BiAb-treated mice showed a significant reduction in their ability to proliferate, and to produce IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha, in response to mTg re-stimulation compared to lymphocytes from untreated mice. Moreover, BiAb-treated mice showed suppressed anti-mTg antibody response, lymphocytic infiltration of the thyroid and follicular destruction. The BiAb targeted to the thyroid most likely facilitated engagement of CTLA-4, resulting in an increase in the number of CD4(+)CD25(+) T cells. These regulatory T cells suppressed in vitro mTg-specific T cell responses, which were associated with an enhanced transforming growth factor (TGF)-beta1 production. Neutralization of TGF-beta1 increased mTg-specific in vitro proliferation of, and IL-2 production by, T cells from BiAb-treated mice. Our data suggest that engagement of CTLA-4 expressed on activated autoreactive T cells in close proximity to the thyroid can increase the number of regulatory T cells and their ability to produce TGF-beta1, with a concomitant reduction in IFN-gamma and TNF-alpha, resulting in suppression of EAT.

Tenascin-C upregulates matrix metalloproteinase-9 in breast cancer cells: direct and synergistic effects with transforming growth factor beta1.

Tenascin-C (TN-C) and matrix metalloproteinases (MMPs) are highly expressed in cancer tissues and probably promote cell migration during cancer progression. TN-C and MMPs are often co-localized in areas of active tissue remodeling in pathologic conditions, suggesting reciprocal regulation. To investigate whether TN-C regulates MMPs expression in cancer cells, we first exposed mammary cancer cells derived from TN-C-deficient mice to TN-C and examined MMPs expression. TN-C was then compared with fibronectin (FN), laminin (LN), basic fibroblast growth factor (b-FGF) and transforming growth factor-beta1 (TGF-beta1). Results of endpoint RT-PCR, quantitative real-time RT-PCR and gelatin zymography demonstrated that TN-C, strongly and dose dependently, upregulates MMP-9 expression in murine mammary cancer cells. TN-C weakly induced MMP-2, MMP-3 and MMP-13. FN and LN induced MMP-9 to lesser extents compared with TN-C. b-FGF had no effect on MMP-9 expression. TGF-beta1 induced MMP-9 expression in a dose-dependent manner, and this induction was significantly enhanced by addition of TN-C. TN-C and TGF-beta1 also upregulated MMP-9 expression in the human breast cancer cell line MDA-MB-231. Neutralization with specific anti-TGF-beta1 antibody showed decreased expression of MMP-9, indicating that TGF-beta controls the baseline MMP-9 expression by a direct autocrine mechanism. Under neutralization of TGF-beta, addition of TN-C still upregulated MMP-9. Conversely, neutralization of endogenous TN-C (in a TN-C-positive mammary cancer cell line) downregulated TGF-beta-induced MMP-9 expression. Thus, TN-C induces MMP-9 expression directly and by collaboration with TGF-beta. These findings reveal a novel role of TN-C in breast cancer progression.

Reversal of tumor-induced immunosuppression by TGF-beta inhibitors.

The immune system is responsible for the early detection and destruction of newly transformed malignant cells. Some transformed cells become immunologically invisible by passive avoidance of immune surveillance (i.e., when tumor cells are immunologically indistinguishable from normal cells). Other transformed cells actively secrete cytokines that effectively blind the immune system to the presence of abnormal antigens on the tumor cell surface. Transforming growth factor-beta ("TGF-beta"), which is expressed by a majority of malignant tumors, is the most potent immunosuppressor and therefore, the most likely cytokine to be responsible for the latter phenomenon. In addition to playing a key role in tumor-induced immunosuppression, TGF-beta stimulates angiogenesis. Interestingly, tumor cells eventually become refractory to TGF-beta-mediated growth arrest, either due to loss of TGF-beta receptors or due to dysregulation in TGF-beta signaling pathways. Neutralization of TGF-beta or inhibition of its production is an effective method of cancer treatment in variety of animal models. Several agents targeting TGF-beta are in the early stages of development and include anti-TGF-beta antibodies, small molecule inhibitors of TGF-beta, Smad inhibitors and antisense gene therapy. Since tumors may express more than one isoform of TGF-beta, these new drugs should target all three TGF-beta isoforms produced by human tumors. The effects of therapies targeting TGF-beta are likely to be synergistic with cytotoxic chemotherapy and immunotherapy. Reversal of TGF-beta-induced immunosuppression is a new and promising approach to cancer therapy, with potential applications in other diseases such as AIDS.

High glucose and endothelial cell growth: novel effects independent of autocrine TGF-beta 1 and hyperosmolarity.

Human endothelial cells were exposed to 5 mM glucose (control), 25 mM (high) glucose, or osmotic control for 72 h. TGF-beta1 production, cell growth, death, and cell cycle progression, and the effects of TGF-beta1 and TGF-beta neutralization on these parameters were studied. High glucose and hyperosmolarity increased endothelial TGF-beta1 secretion (P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducing endothelial cell number (P < 0.001) and increasing cellular protein content (P < 0.001) than the osmotic control. TGF-beta antibody only reversed the antiproliferative and hypertrophic effects of high glucose. High glucose altered cell cycle progression and cyclin-dependent kinase inhibitor expression independently of hyperosmolarity. High glucose increased endothelial cell apoptosis (P < 0.01), whereas hyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF-beta antibody did not reverse the apoptotic effects observed with high glucose. Exogenous TGF-beta1 mimicked the increased S phase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cell cycle progression. These growth effects occurred principally via a TGF-beta1 autocrine pathway. In contrast, apoptosis and endoreduplication occurred independently of this cytokine and hyperosmolarity.