TGF-beta Isoforms Research Articles

Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

BackgroundDuring the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-β isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.MethodsRats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Pseudopregnancy was induced with sex steroids in ovariectomized rats and rats were killed at different days (days 1–9). Uteri were collected and either fixed for immunohistochemical staining (IHC) or processed for RT-PCR and Western analyses. For the in vitro part of the study, rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were purified, cultured and treated with different concentrations of TGF-beta isoforms.ResultsOur results showed that all three TGF-beta isoforms are present, but are localized differently in the endometrium during the estrous cycle and their expression is regulated differently during pseudopregnancy. In cultured stromal cells, we found that TGF-beta3 isoform induced Smad2 phosphorylation, indicating that the Smad pathway is activated by TGF-beta3 in these cells. Furthermore, TGF-beta2 and TGF-beta3 induced a dose-dependant increase of apoptosis in cultured stromal cells, as demonstrated by Hoechst nuclear staining. Noteworthy, TGF-beta2 and TGF-beta3 reduced the level of the anti-apoptotic XIAP protein, as well as the level of phosphorylated/active Akt, a well known survival protein, in a dose-dependent manner.ConclusionThose results suggest that TGF-beta might play an important role in the remodelling endometrium during the estrous cycle and in the regulation of apoptosis in rat decidual cells, in which inhibition of Akt survival pathway might be an important mechanism involved in the regulation of apoptosis.

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF-beta isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF-beta1, -2, and -3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF-beta1 was primarily deposited in the fibrin clot and the unwounded BL. TGF-beta2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF-beta3 was mainly detected in the unwound region of basal epithelial cells. alpha-Smooth muscle actin (alpha-SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, alpha-SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF-beta2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF-beta2 were released into the posterior region of healing stroma on day 14. High levels of alpha-SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF-beta2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF-beta2-mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo.

Deficient activation and different expression of transforming growth factor-beta isoforms in active proliferative diabetic retinopathy and neovascular eye disease.

An increased expression and secretion of angiogenic growth factors was proposed to occur in proliferative diabetic retinopathy and other neovascularizing retinal diseases. However, a loss of anti-angiogenic factors also might promote retinal neovascularization. Therefore we investigated the active and latent vitreous levels of the subtypes of the endothelial anti-mitogen transforming growth factor-beta in vitreous of 58 patients. Four groups of patients were compared: Controls without retinal hypoxia, patients with quiescent and active proliferative diabetic retinopathy (PDR), and patients with severe retinal hypoxia resulting in rubeosis iridis. Whereas the amount of total TGF-beta in the four groups did not differ significantly, latent TGF-beta isoform expression showed complex alterations in ocular vitreous. Levels of active TGF-beta of patients with active PDR (79.5 +/- 28 pg/ml; n = 8) were decreased to 20% of the control levels (378 +/- 55 pg/ml; n = 12; p = 0.0005) and 25% of the mean concentration in quiescent PDR (346 +/- 64 pg/ml; n = 9; p = 0.0021). Levels in rubeosis (52 +/- 10 pg/ml; n = 10) did not differ significantly from those found in active PDR but were decreased to 15% of those in patients with quiescent PDR (p = 0.0004). Furthermore a highly significant inverse correlation between active TGF-beta and alpha2-antiplasmin, a liver produced inhibitor of the activation of TGF-beta by plasmin was noted (r = -0.59; n = 28; p = 0.001). We conclude that deficient activation of TGF-beta occurs in active proliferative diabetic retinopathy and in hypoxic angiogenesis most likely as a consequence of a blood retina barrier breakdown and influx of alpha2-antiplasmin from serum. The disinhibition of endothelial cell proliferation may be a central component in the process of neovascularization.

TGF-β signaling and collagen deposition in chronic rhinosinusitis

Chronic rhinosinusitis is an inflammatory disease with distinct cytokine and remodeling patterns. The objective was to analyze the presence of TGF-beta isoforms, receptors, intracellular signaling, and collagen deposition in chronic rhinosinusitis. Sinonasal mucosal samples obtained from chronic rhinosinusitis with nasal polyps (CRSwNP; n = 13), chronic rhinosinusitis without nasal polyps (CRSsNP; n = 13), and controls (n = 10) were analyzed for TGF-beta isoforms 1 and 2 by means of ELISA and IHC, and for TGF-beta R1, 2, and 3 by RT-PCR and IHC. As downstream proteins, phospho-Smad 2 (pSmad 2) and collagen were analyzed by performing immunostaining and picrosirius red staining, respectively. TGF-beta 1 and 2 protein concentrations, TGF-beta receptor (R) I and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly higher in CRSsNP versus controls. In CRSwNP, TGF-beta 1 protein concentration, TGF-beta RII and TGF-beta RIII mRNA expression, the number of pSmad 2-positive cells, and total collagen amount were significantly lower versus controls. Only TGF-beta 2 protein was found higher in CRSwNP versus controls. A high TGF-beta 1 protein expression, increased TGF-beta RI expression, and a high number of pSmad 2-positive cells all indicate an enhanced TGF-beta signaling in CRSsNP, whereas a low TGF-beta 1 protein concentration, a decreased expression of TGF-beta RII, and a low number of pSmad 2-positive cells in CRSwNP indicate a low level of TGF-beta signaling in CRSwNP. These findings are compatible with the remodeling patterns observed, reflected by a lack of collagen in CRSwNP, and excessive collagen production with thickening of the collagen fibers in the extracellular matrix in CRSsNP.

Adenovirus-mediated gene transfer of fibromodulin inhibits neointimal hyperplasia in an organ culture model of human saphenous vein graft disease

Poor long-term graft patency remains a major limitation of coronary artery bypass grafting using saphenous vein aortocoronary grafts. Neointimal hyperplasia (NIH) represents the principal mechanism of graft failure; a substantial body of evidence implicates transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of NIH. The small leucine-rich proteoglycans decorin and fibromodulin possess TGF-beta-antagonist activity to differing extents and with differing avidities for the isoforms of TGF-beta. We compared their ability to inhibit NIH in an ex vivo model of human saphenous vein organ culture following adenovirus-mediated gene transfer. Surgically prepared human saphenous vein segments received adenovirus expressing fibromodulin (Ad5-Fmod), decorin (Ad5-Dcn), beta-galactosidase (Ad5-lacZ) or vehicle-only. Computerized morphometry 14 days after infection revealed significantly reduced neointimal area, neointimal thickness and intima/media ratio in Ad5-Fmod- and Ad5-Dcn-infected veins. Each parameter was significantly smaller in Ad5-Fmod- than in Ad5-Dcn-exposed segments. Fibrillar collagen content and levels of biologically active TGF-beta were lower in vessels receiving Ad5-Fmod or Ad5-Dcn than in those receiving Ad5-lacZ or vehicle-only. Fibromodulin is a more potent inhibitor of NIH in cultured human saphenous vein than decorin and offers potential therapeutic benefits in saphenous vein graft failure (and possibly in other forms of accelerated atherosclerosis) by reduction of associated neointima formation.

TGF-β/Extracellular Matrix Interactions in Dentin Matrix: A Role in Regulating Sequestration and Protection of Bioactivity

TGF-beta isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin-pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore 3000 system to investigate the binding of TGF-beta isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-beta isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-beta isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-beta isoforms. However, the association with TGF-beta3 was much weaker than that with TGF-beta1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-beta was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-beta isoforms 1 and 3, which may explain why TGF-beta3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-beta1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-beta/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.

Epithelial stem cell‐related alterations in Trichinella spiralis‐infected small intestine

Infection of mice with the parasite Trichinella spiralis leads to small intestinal inflammation, characterized by changes in mucosal architecture and subpopulations of epithelial cells. This model has been used to explore changes in the epithelial proliferative cell population and expression of transforming growth factor-beta (TGF-beta). Histochemical and immunohistochemical studies were undertaken in duodenal samples. Location and number of Ki-67-positive cells were assessed using Score and Wincrypts program. Changes in mRNA transcripts were studied by real-time RT-PCR. T. spiralis infection induced an increase in total number of proliferative (Ki-67-positive) cells per half crypt on day 2 post-infection. Transcription of Math1, a transcription factor required for secretory cell differentiation in the intestine, was up-regulated on days 6-18 post-infection. At these time points, numbers of Paneth cells at the crypt base were also increased and the epithelial proliferative zone was shifted up the crypt-villus axis. Transcription of TGF-beta isoforms within the small intestine was up-regulated on days 6 and 12 post-infection, but anti-TGF-beta antibody treatment had no effect on T. spiralis-induced changes in mucosal architecture or increase in Paneth/intermediate cells. T. spiralis infection promotes an initial increase in small intestinal epithelial proliferation and subsequent cell differentiation along the secretory cell lineage. The resulting increase in numbers of Paneth cells at the crypt base causes the proliferative zone to move up the crypt-villus axis. Further studies are required to determine the significance of an increase in the expression of TGF-beta transcripts.

Increased blood plasma concentrations of TGF‐β1 and TGF‐β2 after treatment with intravenous immunoglobulins in childhood autoimmune diseases

Transforming growth factor-beta (TGF-beta), a multifunctional, immunosuppressive cytokine, is shown to be present in substantial amounts in commercially available intravenous immunoglobulin (IVIG) preparations. To assess whether TGF-beta isoforms are changed in the plasma of paediatric patients with childhood autoimmune diseases after IVIG infusion, 17 patients who received over a period of 12 months overall 56 IVIG infusions (Endobulin) were enrolled in a study. High levels of TGF-beta1 (16.95 +/- 8.16 ng/ml) as well as TGF-beta2 (62.71 +/- 9.50 ng/ml) were detected in the used 56 IVIG probes. TGF-beta1 and TGF-beta2 plasma concentrations were measured prior and 120 min after IVIG infusions by specific TGF-beta ELISA. Interestingly, significant increased TGF-beta1 and TGF-beta2 plasma levels were found in patients after treatment with IVIG. This data suggest that a TGF-beta-mediated mechanism of action may accompany other molecular effects of IVIG therapy. The amount of the potent anti-inflammatory TGF-beta isoforms within the IVIG preparations may exert a differentiated view regarding the manifold indications of IVIG therapy.

Mice with disrupted TGFbeta signaling have normal cerebella development, but exhibit facial dysmorphogenesis and strain-dependent deficits in their body wall.

The transforming growth factor betas (TGFbetas) are context-dependent regulators of neurons in vitro, but their physiological functions in the brain are unclear. Haploinsufficiency of either Tgfbeta1 or Tgfbeta2 leads to age-related deterioration of neurons, but the development of the brain is normal in the full absence of either of these genes. However, some individuals with mis-sense mutations of TGFbeta receptors are mentally retarded, suggesting that the TGFbeta isoforms can compensate for each other during brain development. This possibility was tested by generating mice (NSE x PTR) with neuron-specific expression of a dominant-negative inhibitor of TGFbeta signaling. The NSE x PTR mice with a FVBxC57Bl/6 genetic background were viable and developed normally despite strong neuronal expression of the inhibitor of TGFbeta signaling. Their cerebella were of normal size and contained normal numbers of neurons. When the genetic background of the mice was changed to C57BL/6, the phenotype of the mice became neonatal lethal, with the neonates exhibiting various malformations. The malformations correlated with sites of non-neuronal expression of the transgenes and included facial dysmorphogenesis, incomplete closure of the ventral body wall and absence of intestinal motility. The C57BL/6 Tgfbm1-3 alleles, which modulate the phenotype of Tgfbeta1(-/-) mice, were not major determinants of the NSE x PTR phenotype. The data suggest that the development of the cerebellum is insensitive to the level of TGFbeta signaling, although this may be dependent on the genetic background.

TβR-II Discriminates the High- and Low-Affinity TGF-β Isoforms via Two Hydrogen-Bonded Ion Pairs

The TGF-beta isoforms, TGF-beta1, -beta2, and -beta3, share greater than 70% sequence identity and are almost structurally identical. TGF-beta2 differs from the others, however, in that it binds the TGF-beta type II receptor (TbetaR-II) with much lower affinity than either TGF-beta1 or -beta3. It has been previously shown that three conserved interfacial residues, Arg25, Val92, Arg94, in TGF-beta1 and -beta3 are responsible for their high-affinity interaction with TbetaR-II. In this study, the role of each of these residues was examined by creating single, double, and triple substitutions resulting in both TGF-beta3 loss-of-function and TGF-beta2 gain-of-function variants. One-dimensional 1H NMR spectra of the variants confirmed a lack of large structural perturbations. Affinities, kinetics, and thermodynamics for TbetaR-II binding were determined by surface plasmon resonance biosensor analysis. Double substitutions revealed that nearly all of the high-affinity binding is contributed by Arg25 and Arg94. Single site substitutions showed that Arg94 makes the greatest contribution. Substitution of Arg25 and Arg94 with alanine verified the requirement of the arginine guanidinium functional groups for the highly specific hydrogen-bonded ion pairs formed between Arg25 and Arg94 of TGF-beta1 and -beta3, and Glu119 and Asp32 of TbetaR-II. Further kinetic and thermodynamic analyses confirmed that Arg25 and Arg94 are primarily responsible for high-affinity binding and also revealed that noninterfacial longer range effects emanating from the TGF-beta structural framework contribute slightly to TbetaR-II binding. Growth inhibition assays showed that binding changes generally correlate directly with changes in function; however, a role Val92 in this cellular context was uncovered.

Bone tissue content of TGF-β2 changes with time in human heterotopic ossification after total hip arthroplasty

Transforming growth factor beta isoforms (TGF-β1, TGF-β2, and TGF-β3) most likely play a role in bone physiology, but little is known about their relative importance in normal as well as in heterotopic bone. This study focused on possible differences in the localization and relative content of different TGF beta isoforms in heterotopic ossifications (HO) by comparing HOs, which have developed less than 17 months (immature HOs) with those developed 3–9 years (mature HOs). The HOs were harvested after total hip arthroplasty (THA) during revision surgery. The HO samples were decalcified, embedded in paraffin and sectioned. Azan staining was used to evaluate histological structure of the ossifications and immunohistochemical analysis was performed to estimate the localization of three TGF beta isoforms in the HOs. Comparison of different TGF beta isoforms in the immature and the mature ossifications showed that the content of TGF-β2 was decreased by almost three times in the mature HO as compared to the immature HO (p = 0.0064). The proportions of other isoforms in HOs did not differ significantly. This study shows that the relative importance of TGF betas change with HO development.

Downregulation of SMAD2, 4 and 6 mRNA and TGFβ Receptor I mRNA in Lesional and Non-lesional Psoriatic Skin

Transforming growth factor beta (TGFbeta) has been suggested to be an effective inhibitor of the increased keratinocyte proliferation in psoriasis. Three TGFbeta isoforms are described (TGFbeta1, 2 and 3), signalling via a heteromeric receptor complex of TGFbetaRI and TGFbetaRII. Receptor binding activates SMAD2, 3 and 4, which translocate into the nucleus and regulate TGFbeta-responsive genes. SMAD6 and 7 proteins represent a negative feedback loop inhibiting the TGFbeta-SMAD signalling path-way. As TGFbeta1 overexpression inhibits keratinocyte proliferation, the aim of this study was to investigate with real-time RT-PCR the expression of TGFbeta1, 2 and 3, TGFbetaRI and TGFbetaRII and SMAD2, 3, 4, 6 and 7 in lesional and non-lesional psoriatic skin from 13 patients with chronic plaque-type psoriasis as compared to skin from 10 healthy subjects . The study data demonstrate significantly downregulated TGFbetaRI and SMAD2, 4 and 6 mRNA expression in lesional and non-lesional psoriatic skin. SMAD7 mRNA expression was significantly decreased in lesional psoriatic skin compared with both non-lesional psoriatic skin and healthy skin. A significant TGFbeta3 and TGFbetaRII mRNA upregulation exclusively in non-lesional psoriatic skin but no significant difference in the expression of TGFbeta1 and 2 was found. The results of this study suggest that the expression of TGFbeta isoforms, receptors and SMADs may be involved in the increased proliferation of keratinocytes in psoriatic skin.

Hepatic nephroblastoma overexpressed (NOV/CCN3) in experimental liver fibrosis models

NOV/CCN3 is a matricellular protein of the CCN family, comprising CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). CCN proteins are involved in mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration of multiple cell types. Compared with CTGF, NOV has been studied to a lesser degree and its biological role in liver fibrosis remains primarily unknown. We therefore investigated hepatic expression of NOV in chronic liver injury upon long term CCl4 treatment and bile duct ligation (BDL) rat models. NOV gene expression was quantified by TaqMan RT-PCR, immunohistochemistry and ELISA. In both models of chronic liver injury, NOV mRNA showed significant and sustained upregulation. Specifically in BDL models, immunohistochemistry revealed clear expression of NOV protein in portal myofibroblasts, HSC and proliferative bile ducts along the fibrotic septa. Additionally, CCL4 models showed positive staining in particular regenerative hepatocytes. By contrast, TaqMan RT-PCR demonstrated no NOV expression in cultured hepatocytes isolated from naive and CCl4 treated rats. Culture-activated HSC however showed significant upregulation of NOV, reaching the highest levels in MFB. Upon HSC stimulation with TGF-beta and PDGF isoforms, only PDGF-B and –D (known to induce HSC proliferation) significantly reduced NOV expression in both mRNA and protein levels (Western blot and ELISA) while TGF-beta showed but modest effects. Since NOV forms physiological complexes with fibulin-1 in blood, we set to analyze rat serum NOV through ELISA, finding a serum concentration of 5–10 ng/ml but no significant difference between the control and BDL rats. The indifference found in serum concentration may result from NOV being produced by different organs, specifically adrenal cortex, and secreted into the blood circulation. Conclusion: NOV appears to play its role in liver fibrogenesis, the functionality of which merits to be further established.

Differential Effects of TGF-β1 and TGF-β3 on Chondrogenesis in Posterofrontal Cranial Suture–Derived Mesenchymal Cells In Vitro

Transforming growth factor (TGF)-beta1 has been associated with cranial suture fusion, whereas TGF-beta3 has been associated with suture patency. The mouse posterofrontal suture, analogous to the human metopic suture, fuses through endochondral ossification. TGF-beta1 and TGF-beta3 expression in the posterofrontal suture was examined by immunohistochemistry. Next, the authors established cultures of suture-derived mesenchymal cells from the posterofrontal suture and examined the cellular responses to TGF-beta1 and TGF-beta3. Proliferation in response to TGF-beta isoforms was examined by bromodeoxyuridine incorporation. High-density micromass culture of posterofrontal mesenchymal cells was used to study the effect of TGF-beta1 and TGF-beta3 on chondrogenic differentiation. TGF-beta1 but not TGF-beta3 protein was highly expressed in chondrocytes within the posterofrontal suture. Significant increases in posterofrontal cell proliferation were observed with TGF-beta3 but not TGF-beta1. TGF-beta1 led to significant increases in chondrogenic-specific gene expression (including Sox9, Col II, Aggrecan, and Col X) as compared with moderate effects of TGF-beta3. TGF-beta1 increased cellular adhesion molecule expression (N-cadherin and fibronectin) and promoted cellular condensation, whereas TGF-beta3 increased cellular proliferation (PCNA expression). Finally, TGF-beta1 and, to a lesser extent, TGF-beta3 induced the expression of fibroblast growth factors (FGF-2 and FGF-18). TGF-beta1 and TGF-beta3 exhibit marked differences in their effects on chondrogenesis in posterfrontal suture-derived mesenchymal cells, influencing different stages of chondrogenic differentiation. TGF-beta3 significantly increased cellular proliferation, whereas TGF-beta1 induced precartilage condensation, promoting chondrocyte differentiation.

Transforming growth factor-β1 is the predominant isoform required for breast cancer cell outgrowth in bone

Transforming growth factor (TGF)-beta signaling is a potent modulator of the invasive and metastatic behavior of breast cancer cells. Indeed, breast tumor responsiveness to TGF-beta is important for the development of osteolytic bone metastases. However, the specific TGF-beta isoforms that promote breast cancer outgrowth in bone is unknown. We demonstrate that expression of a TGF-beta ligand trap, which neutralizes TGF-beta1 and TGF-beta3, in MDA-MB-231 breast cancer cells diminished their outgrowth in bone and reduced the severity of osteolytic lesion formation when compared with controls. We further show that a reduction or loss of TGF-beta1 expression within the bone microenvironment of TGF-beta1+/- and TGF-beta1-/- mice significantly reduced the incidence of breast tumor outgrowth compared with wild-type animals. Interestingly, those tumors capable of growing within the tibiae of TGF-beta1-deficient mice had upregulated expression of all three TGF-beta isoforms. Finally, breast cancer cells expressing the TGF-beta ligand trap showed a pronounced reduction in their ability to form osteolytic lesions when injected into the tibiae of TGF-beta1+/- mice. Thus, our studies show that both host- and tumor-derived TGF-beta expression plays a critical role during the establishment and outgrowth of breast cancer cells in bone.

Dual functions for LTBP in lung development: LTBP‐4 independently modulates elastogenesis and TGF‐β activity

The latent TGF-beta binding proteins (LTBP) -1, -3, and -4 are extracellular proteins that assist in the secretion and localization of latent TGF-beta. The null mutation of LTBP-4S in mice causes defects in the differentiation of terminal air-sacs, fragmented elastin, and colon carcinomas. We investigated lung development from embryonic day 14.5 (E14.5) to day 7 after birth (P7) in order to determine when the defects in elastin organization initiate and to further examine the relation of TGF-beta signaling levels and air-sac septation in Ltbp4S-/- lungs. We found that defects in elastogenesis are visible as early as E14.5 and are maintained in the alveolar walls, in blood vessel media, and subjacent airway epithelium. The air-sac septation defect was associated with excessive TGF-beta signaling and was reversed by lowering TGF-beta2 levels. Thus, the phenotype is not directly reflective of a change in TGF-beta1, the only TGF-beta isoform known to complex with LTBP-4. Reversal of the air-sac septation defect was not associated with normalization of the elastogenesis indicating two separate functions of LTBP-4 as a regulator of elastic fiber assembly and TGF-beta levels in lungs.

Localization of small leucine‐rich proteoglycans and transforming growth factor‐β in human oral mucosal wound healing

Wound healing in oral mucosa is fast and results in little scar formation as compared with skin. The biological mechanisms underlying this property are poorly understood but may provide valuable information about the factors that promote wound regeneration. Small leucine-rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are extracellular matrix molecules that regulate collagen fibrillogenesis, inhibit transforming growth factor-beta (TGF-beta) activity and reduce scarring. In the present study, we analyzed accumulation of SLRPs and TGF-beta during non-scarring human oral mucosal wound healing. Biopsies were collected from healthy volunteers from unwounded tissue and from standardized experimental wounds 3-60 days postwounding. Localization of SLRPs, TGF-beta1 and TGF-beta3 was analyzed by immunohistochemical staining and quantitated by image analysis. Double immunostaining was used to study localization of SLRPs or active TGF-beta in distinct cells. Decorin, biglycan, fibromodulin, and TGF-beta isoforms showed significantly increased accumulation in the wound extracellular matrix and distinct wound cells while the abundance of lumican in the extracellular matrix was strongly reduced during wound healing. Localization and abundance of fibromodulin, lumican, and TGF-beta isoforms was also spatiotemporally regulated in the wound epithelium. The findings suggest that SLRPs regulate wound reepithelialization and connective tissue regeneration during oral mucosal wound healing.

Pro- and anti-fibrotic effects of TGF- in scleroderma

The University of California at Davis 200 and 206 (UCD-200/206) lines of chickens have proven to be the animal model that best reflects the situation in human SSc. We have demonstrated a misbalance of pro-fibrotic (TGF-beta1) and anti-fibrotic (TGF-beta2 and -beta3) TGF-beta isoforms as a possible cause for fibrotic alterations in this model. This opens new avenues for diagnosis and therapy for this still intractable condition.

Studies of TGF-β 1-3 in Serosal Fluid During Abdominal Surgery and Their Effect on In Vitro Human Mesothelial Cell Proliferation

Increased transforming growth factor-beta (TGF-beta) levels are associated with fibrosis, affected cell proliferation, and postsurgical adhesion development, but the knowledge regarding TGF-beta response to the surgical trauma is limited. This study investigated TGF-beta(1-3) isoforms and fibrinolytical factors in peritoneal serosal fluid during abdominal surgery, together with the in vitro effect of TGF-beta(1-3) on human mesothelial cell proliferation. Total as well as biologically active TGF-beta(1-3) and fibrinolytic factors: t-PA, uPA, and PAI-1 were measured in serosal fluid and plasma from 23 patients undergoing colorectal cancer surgery. In vitro proliferation of human primary mesothelial cell cultures upon TGF-beta(1-3) stimulation was also investigated. Total TGF-beta1 and TGF-beta2 levels were similar in serosal fluid and plasma while active fractions were increased in serosal fluid. In contrast, total fraction of TGF-beta3 was higher in serosal fluid compared with plasma, while levels of active fractions did not differ. Plasminogen activators (t-PA, uPA) were elevated while the inhibitor (PAI-1) was decreased in serosal fluid compared with plasma. The in vitro mesothelial cell proliferation studies revealed that high TGF-beta(1-3) concentrations decreased cell proliferation, while lower concentrations of TGF-beta1 increased mesothelial cell proliferation. This human study shows increased active TGF-beta levels in peritoneal serosal fluid, compared with plasma, during abdominal surgery and that TGF-beta1 at physiological concentrations increased human mesothelial cell proliferation in vitro. TGF-beta cytokines may be involved in postsurgical adhesion formation.

Expression, purification and characterization of BGERII: a novel pan-TGF inhibitor

Transforming growth factor beta (TGFbeta) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFbeta isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFbeta type II (RII) and betaglycan (BG) have been utilized as TGFbeta inhibitors, their antagonistic potency against different TGFbeta isoforms varies considerably because RII does not appreciably bind to TGFbeta2 whereas BG binds weakly to TGFbeta1 and TGFbeta3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BG(E)) and the extracellular domain of RII. The fusion protein (named BG(E)RII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFbeta1 and TGFbeta3 than a commercially available soluble RII and to TGFbeta2 than a commercially available soluble BG. More significantly, whereas BG(E) or RII alone showed no antagonistic activity towards TGFbeta2, BG(E)RII inhibited the signaling of both TGFbeta1 and TGFbeta2 in cell-based assays including TGFbeta-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFbeta-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BG(E)RII was found to have greater activity than other potent TGFbeta inhibitors in blocking the signaling of TGFbeta1 and TGFbeta3. Thus, BG(E)RII is a potent pan-TGFbeta inhibitor in vitro and has potential for blocking TGFbeta-induced pathogenesis in vivo.