Presence Of TGF-beta Research Articles

Mechanisms of tissue inhibitor of metalloproteinase 1 augmentation by IL-13 on TGF-β1–stimulated primary human fibroblasts

TGF-beta induces expression of tissue inhibitor of metalloproteinase 1 (TIMP-1), a potent inhibitor of matrix metalloproteinases that controls extracellular matrix metabolism and deposition. IL-13 alone does not induce TIMP-1, but in combination with TGF-beta it augments TIMP-1 expression. Although these interactions have implications for remodeling in asthma, little is understood regarding the mechanisms controlling TIMP-1 product. To explore the role of Smads and mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in the TIMP-1 augmentation by IL-13+TGF-beta1 in primary human airway fibroblasts. Real-time PCR, Western blot, ELISA, and transient transfection were used to evaluate the mechanisms of TIMP-1 augmentation. IL-13 enhanced TGF-beta1-induced Smad-2 and Smad-3 phosphorylation, transient transfection with dominant-negative Smad-2 or Smad-3 decreased TIMP-1 mRNA expression in the presence of TGF-beta1 and IL-13+TGF-beta1 through inhibition of Smad-2 or Smad-3 phosphorylation. ERK phosphorylation was increased by IL-13 and IL-13+TGF-beta1. MEK-ERK inhibition decreased TIMP-1 mRNA/protein to a greater degree after IL-13+TGF-beta1 stimulation versus TGF-beta1 alone. MEK-ERK inhibition also significantly increased Akt phosphorylation under all conditions and decreased Smad-3 phosphorylation in the presence of IL-13+TGF-beta1. In contrast, phosphoinositide-3 kinase-Akt inhibition increased phosphorylation of ERK and Smads, leading to increased TIMP-1. These results indicate that IL-13 augments TGF-beta1-induced TIMP-1 expression through increased Smad phosphorylation. These increases occur as TGF-beta1 downregulates IL-13-induced phosphoinositide-3 kinase activation while leaving the positive effect of IL-13-induced ERK on Smad signaling. This augmentation of TGF-beta1-induced TIMP-1 by IL-13 could contribute to the fibrosis and airway remodeling seen in the presence of T(H)2 inflammation in asthma.

CD28 Regulates de novo Generation of Tregs through IL-2 in the Periphery (88.49)

Abstract CD28 plays a critical role in Treg development in the thymus and also contributes to Treg survival and homeostasis in the periphery. However, the role of CD28 in the differentiation of Tregs (CD4+Foxp3+) from naïve T cells (CD4+Foxp3-) in the periphery is unclear. To address this question, we stimulated naïve T cells (CD4+CD25-) with APCs plus anti-CD3 mAb in the presence of TGF-beta. We found that differentiation of Tregs was essentially blocked when CD28 costimulation was absent either by using CD28−/− T cells or B7−/− APCs in the culture. The requirement of CD28 for Treg differentiation was further confirmed in vivo, where WT or CD28−/− naïve T cells were adoptively transferred into irradiated syngeneic or allogeneic recipient. Using CFSE labeling, we found that differentiation of Tregs from WT naïve T cells was irrelevant to the number of cell division, and even T cells without division could also be “converted” into Tregs. Naïve T cells without CD28 divided much more slowly and did not differentiate into Tregs regardless of cell division. The requirement of CD28 for Treg differentiation was mediated through IL-2, in that neutralization of IL-2 with its specific antibody blocked Treg differentiation from WT naïve T cells and additional IL-2 restored Treg differentiation from CD28−/− naïve T cells. We further asked whether other common gamma-chain cytokines have a similar role as IL-2, and found that IL-7, but not IL-4 or IL-15, could partially restored Treg differentiation in the replacement of IL-2. In conclusion, our current study indicates that CD28 is required for Treg differentiation from naïve T cells through IL-2 production in the periphery.

Expression of interferon-gamma (IFN-gamma), IL-10, IL-12 and transforming growth factor-beta (TGF-beta) mRNA in synovial fluid cells from patients in the early and late phases of rheumatoid arthritis (RA).

The expression of immunoregulatory cytokines was investigated in freshly isolated synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with RA, using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. IFN-gamma, TGF-beta, IL-10 and IL-12 (p40) transcripts were detected in SFMC of patients with early disease (<1 year duration) as well as in patients with long standing arthritis (>1 year). The expression of IFN-gamma, IL-10 and IL-12 mRNA was increased in SFMC compared with RA PBMC. In addition, the expression was higher in RA SFMC than in PBMC from health control individuals. Immunoassay analysis of the secreted IL-12 heterodimer demonstrated increased levels in RA SF compared with levels found in serum from RA patients and control individuals. High levels of TGF-beta mRNA were found in SFMC, but a significantly decreased TGF-beta/beta2-microglobulin (beta2-M) ratio was found compared with PBMC from both patients and control individuals. IL-4mRNA could not be detected, either in SFMC or in PBMC. Cytokine expression in RA PBMC did not differ from control PBMC, with the exception of a decreased TGF-beta/beta2-M ratio in RA patients with early disease. Our findings of IFN-gamma mRNA and IL-12, but undetectable levels of IL-4 mRNA, suggest that the synovitis is characterized by a type 1 immune response. The presence of TGF-beta and IL-10 mRNA indicates that immunosuppressive cytokines may also operate in the inflamed joint, although their level of expression may not be sufficient for down-modulation of immune activation.

Involvement of transforming growth factor-β in the expression of gicerin, a cell adhesion molecule, in the regeneration of hepatocytes

Gicerin, an Ig-superfamily cell adhesion molecule, appears transiently in embryonic tissues including those of the nervous, urogenital, respiratory and digestive systems, and it promotes neurite extension, cell migration and epithelialization through its cell adhesive activities. In addition, gicerin also reappears in regenerating tissue after suffering either a traumatic injury or a viral infection. In the present study, we examined the expression pattern of gicerin in the regeneration of hepatocytes. Immunohistochemically, gicerin protein appeared in the regenerating hepatocytes of carbon tetrachloride (CCl4)-induced acute hepatitis, while it was scarcely expressed in the hepatocytes of normal mouse liver. Real-time PCR revealed the up-regulation of gicerin transcription in the regenerating process of CCl4-induced hepatitis. The expression of transforming growth factor (TGF)-beta1 was also increased during the regeneration. Furthermore, the gicerin mRNA expression increased during the process of an in vitro hepatocyte regeneration model using mouse primary hepatocytes and hepa 1-6 cells. To note, the mRNA levels of gicerin in these cells were enhanced by the presence of TGF-beta1. Collectively, these findings suggest that TGF-beta1 may therefore regulate gicerin expression in hepatocytes leading to liver regeneration by cell-cell or cell-ECM interactions.

Growth regulation of simian and human AIDS-related non-Hodgkin's lymphoma cell lines by TGF-β1 and IL-6

BackgroundAIDS-related non-Hodgkin's lymphoma (AIDS-NHL) is the second most frequent cancer associated with AIDS, and is a frequent cause of death in HIV-infected individuals. Experimental analysis of AIDS-NHL has been facilitated by the availability of an excellent animal model, i.e., simian Acquired Immunodeficiency Syndrome (SAIDS) in the rhesus macaque consequent to infection with simian immunodeficiency virus. A recent study of SAIDS-NHL demonstrated a lymphoma-derived cell line to be sensitive to the growth inhibitory effects of the ubiquitous cytokine, transforming growth factor-beta (TGF-beta). The authors concluded that TGF-beta acts as a negative growth regulator of the lymphoma-derived cell line and, potentially, as an inhibitory factor in the regulatory network of AIDS-related lymphomagenesis. The present study was conducted to assess whether other SAIDS-NHL and AIDS-NHL cell lines are similarly sensitive to the growth inhibitory effects of TGF-beta, and to test the hypothesis that interleukin-6 (IL-6) may represent a counteracting positive influence in their growth regulation.MethodsGrowth stimulation or inhibition in response to cytokine treatment was quantified using trypan blue exclusion or colorimetric MTT assay. Intracellular flow cytometry was used to analyze the activation of signaling pathways and to examine the expression of anti-apoptotic proteins and distinguishing hallmarks of AIDS-NHL subclass. Apoptosis was quantified by flow cytometric analysis of cell populations with sub-G1 DNA content and by measuring activated caspase-3.ResultsResults confirmed the sensitivity of LCL8664, an immunoblastic SAIDS-NHL cell line, to TGF-beta1-mediated growth inhibition, and further demonstrated the partial rescue by simultaneous treatment with IL-6. IL-6 was shown to activate STAT3, even in the presence of TGF-beta1, and thereby to activate proliferative and anti-apoptotic pathways. By comparison, human AIDS-NHL cell lines differed in their responsiveness to TGF-beta1 and IL-6. Analysis of a recently derived AIDS-NHL cell line, UMCL01-101, indicated that it represents immunoblastic AIDS-DLCBL. Like LCL-8664, UMCL01-101 was sensitive to TGF-beta1-mediated inhibition, rescued partially by IL-6, and demonstrated rapid STAT3 activation following IL-6 treatment even in the presence of TGF-beta1.ConclusionThese studies indicate that the sensitivity of immunoblastic AIDS- or SAIDS-DLBCL to TGF-beta1-mediated growth inhibition may be overcome through the stimulation of proliferative and anti-apoptotic signals by IL-6, particularly through the rapid activation of STAT3.

Characterization of novel transforming growth factor-beta type I receptors found in malignant pleural effusion tumor cells

BackgroundTumors expressing a transforming growth factor-beta type I receptor (TβRI) mutant with sequence deletions in a nine-alanine (9A) stretch of the signal peptide are reported to be highly associated with disease progression. Expression of this mutant could interfere with endogenous TGFβ signaling in the cell. However, little is known about the importance of the remaining part of the signal peptide on the cellular function of TβRI.ResultsWe cloned and identified four new in-frame deletion variants of TβRI, designated DM1 to DM4, in pleural effusion-derived tumor cells. Intriguingly, DM1 and DM2, with a small region truncated in the putative signal peptide of TβRI, had a serious defect in their protein expression compared with that of the wild-type receptor. Using serial deletion mutagenesis, we characterized a region encoded by nucleotides 16–51 as a key element controlling TβRI protein expression. Consistently, both DM1 and DM2 have this peptide deleted. Experiments using cycloheximde and MG132 further confirmed its indispensable role for the protein stability of TβRI. In contrast, truncation of the 9A-stretch itself or a region downstream to the stretch barely affected TβRI expression. However, variants lacking a region C-terminal to the stretch completely lost their capability to conduct TGFβ-induced transcriptional activation. Intriguingly, expression of DM3 in a cell sensitive to TGFβ made it significantly refractory to TGFβ-mediated growth inhibition. The effect of DM3 was to ablate the apoptotic event induced by TGFβ.ConclusionWe identified four new transcript variants of TβRI in malignant effusion tumor cells and characterized two key elements controlling its protein stability and transcriptional activation. Expression of one of variants bestowed cancer cells with a growth advantage in the presence of TGFβ. These results highlight the potential roles of some naturally occurring TβRI variants on the promotion of tumor malignancy.

Transforming growth factor-β1 suppresses hepatitis B virus replication primarily through transcriptional inhibition of pregenomic RNA

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with pivotal roles in the regulation of cellular functions and immune responses. In this study, we found that TGF-beta1 was able to effectively suppress hepatitis B virus (HBV) replication. In the presence of TGF-beta1, the level of viral replicative intermediates was dramatically decreased, both in actively dividing cells and in confluent cells. At the same time, the levels of viral transcripts, core protein, and nucleocapsid were significantly diminished by TGF-beta1 treatment. Interestingly, the inhibitory activity of TGF-beta1 was associated with preferential reduction of the level of pregenomic RNA compared with pre-C mRNA. Further analysis indicated that TGF-beta1 might exert its antiviral effect primarily through reducing expression of the HBV core protein by transcriptional regulation instead of posttranscriptional modification. TGF-beta1 may play a dual role in HBV infection, in the suppression of immune responses against viral infection and in the direct inhibition of viral replication, resulting in minimization of liver damage in patients with chronic hepatitis.

Immunohistochemical Examination of PDGF-AB, TGF-.BETA. and their Receptors in the Hemocytes of a Tick, Ornithodoros moubata (Acari: Argasidae)

Growth factors, Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor (TGF)-beta, were demonstrated in vertebrate and invertebrate immmunocytes. It is generally known that the growth factors are important in various biological processes, such as the regulation of cell differentiation, development and wound healing. In the present study, the presence of TGF-beta1 and PDGF-receptor-alpha in plasmatocytes and PDGF-AB in granulocytes of a soft tick, Ornithodoros moubata, was confirmed immunohistochemically. The tick midgut might be damaged by intracellular digestion and penetration of protozoa. Therefore, it is considered that PDGF from granulocytes may affect the PDGF-receptor-alpha in plasmatocytes and TGF-beta from plasmatocytes may function to repair the midgut. The results obtained here add to the elucidation of the functions of tick hemocytes.

Essential function of HIPK2 in TGFβ-dependent survival of midbrain dopamine neurons

Transforming growth factor beta (TGFbeta) is a potent trophic factor for midbrain dopamine (DA) neurons, but its in vivo function and signaling mechanisms are not entirely understood. We show that the transcriptional cofactor homeodomain interacting protein kinase 2 (HIPK2) is required for the TGFbeta-mediated survival of mouse DA neurons. The targeted deletion of Hipk2 has no deleterious effect on the neurogenesis of DA neurons, but leads to a selective loss of these neurons that is due to increased apoptosis during programmed cell death. As a consequence, Hipk2(-/-) mutants show an array of psychomotor abnormalities. The function of HIPK2 depends on its interaction with receptor-regulated Smads to activate TGFbeta target genes. In support of this notion, DA neurons from Hipk2(-/-) mutants fail to survive in the presence of TGFbeta3 and Tgfbeta3(-/-) mutants show DA neuron abnormalities similar to those seen in Hipk2(-/-) mutants. These data underscore the importance of the TGFbeta-Smad-HIPK2 pathway in the survival of DA neurons and its potential as a therapeutic target for promoting DA neuron survival during neurodegeneration.

Toll-like receptor triggered dendritic cell maturation and IL-12 secretion are necessary to overcome T-cell inhibition by glioma-associated TGF-β2

Malignant gliomas are able to secrete large amounts of immunosuppressive cytokines like transforming growth factor beta 2 (TGF-beta2) and regularly escape from immune surveillance. Many strategies have been developed to induce potent anti-glioma responses, among those the use of dendritic cells (DC) as therapeutic vaccines. Here, we report that both mature DC and IL-12 secretion are necessary to overcome T-cell inhibition by TGF-beta2. Flow cytometric analyses showed that TGF-beta2 does not suppress the upregulation of MHC (major histocompatibility complex) class II molecules and the T cell stimulatory capacity of human DC that were stimulated with a strong cytokine cocktail containing tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-6 and prostaglandin E2 (PGE2). Moreover, TGF-beta2 signaling studies revealed that these cytokine-matured DC become unresponsive to TGF-beta2. Although both mature and immature DC expressed comparable amounts of the TGF-beta receptor type II, Smad2 phosphorylation and subsequent upregulation of Smad7 was inhibited in mature DC, but not immature DC. However, further analysis revealed that mature DC alone are not sufficient to mediate full T cell activation in the presence of TGF-beta2, unless IL-12 is added to the DC/T-cell coculture. Finally, we demonstrate that MHC class II expression and IL-12 secretion by DC are not disturbed by TGF-beta2 after DC stimulation with a modified maturation cocktail containing the Toll-like receptor (TLR)-ligands Poly I:C or R848, TNF-alpha, IL-1beta and INF-gamma. These data imply that mature DC retaining their capacity to produce IL-12 are of favorable use in glioma immunotherapy and suggest that TLR triggering of DC plays an important role to elicit a strong immune response in the immunosuppressive environment of malignant gliomas.

Increased Prevalence of Regulatory T Cells (Treg) is Induced by Pancreas Adenocarcinoma

We reported earlier that patients with breast or pancreas cancer have an increased prevalence of regulatory T cells (Treg) in the blood and tumor draining lymph nodes (TDLNs) compared with healthy individuals. In the current study, we tested the hypothesis that tumor cells promote the prevalence of Treg. The transforming growth factor-beta (TGF-beta) secreting murine pancreas adenocarcinoma, Pan02 cell line was injected into syngeneic C57BL/6 mice and the prevalence of Treg in the TDLNs and tumor spleen was measured weekly. Compared with control mice, the prevalence of CD25+ CD4+ cells in TDLNs and in tumor spleen increased with tumor growth. Analysis of these CD25+ CD4+ T cells in vitro confirmed expression of the Treg marker, Foxp3. In addition, their functional activity resembled that of Treg, as evidenced by a poor proliferative capacity; suppression of proliferation of CD25- CD4 or CD8T cells and inhibition of interferon-gamma release by CD25- CD4+ T cells. Reconstitution of Pan02-bearing Rag-/- mice with naive syngeneic CD25- CD4+ T cells induced CD25+ CD4+ Foxp3+ T cells in TDLNs, but not in the spleen. In contrast, Foxp3 was not detected in unreconstituted Pan02-bearing Rag-/- mice, or reconstituted mice bearing a TGF-beta-negative esophageal tumor. Furthermore, administration of neutralizing anti-TGF-beta antibody blocked the induction of Foxp3 in reconstituted Pan02-bearing Rag-/- mice. These results mimic earlier in vitro studies showing induction of Foxp3 through CD3 plus CD28 stimulation in the presence of TGF-beta. We conclude that Pan02 tumor promotes the prevalence of Treg, in part through the secretion of TGF-beta, which may result in immune evasion.

Dose- and Time-Dependent Effects of Cyclic Hydrostatic Pressure on Transforming Growth Factor-β3-Induced Chondrogenesis by Adult Human Mesenchymal Stem Cellsin Vitro

This study examined effects of varying magnitudes of intermittent hydrostatic pressure (IHP) applied for different times on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. hMSCs were exposed to 0.1, 1, and 10 MPa of IHP at a frequency of 1 Hz for 4 h/day for 3, 7, and 14 days in the presence of transforming growth factor (TGF-beta3). Chondrogenesis was characterized by gene expression, macromolecule production, and extracellular matrix deposition. Exposure of hMSCs to 0.1 MPa of IHP increased SOX9 and aggrecan mRNA expression by 2.2- and 5.6-fold, respectively, whereas type II collagen mRNA expression responded maximally at 10 MPa. Production of sulfated glycosaminoglycan responded to IHP of 1 MPa and 10 MPa, whereas collagen levels increased only at 10 MPa. Morphologically, matrix condensation occurred with increased IHP, concomitant with collagen expression. This study demonstrated that different levels of IHP differentially modulate hMSC chondrogenesis in the presence of TGF-beta3. The data suggest that tissue engineering of articular cartilage through application or recruitment of hMSCs can be facilitated by mechanical stimulation.

Effects of Hydrostatic Pressure and Transforming Growth Factor-β3 on Adult Human Mesenchymal Stem Cell Chondrogenesis In Vitro

This study examined the effects of intermittent hydrostatic pressure (IHP) and transforming growth factor-beta 3 on chondrogenesis of adult human mesenchymal stem cells (hMSCs) in vitro. Chondrogenic gene expression was determined by quantifying mRNA signal levels for SOX9, a transcription factor critical for cartilage development and the cartilage matrix proteins, aggrecan and type II collagen. Extracellular matrix production was determined by weight and histology. IHP was applied to hMSCs in pellet culture at a level of 10 MPa and a frequency of 1 Hz for 4 h per day for periods of 3, 7, and 14 days. hMSCs responded to addition of TGF-beta 3 (10 ng/mL) with a greater than 10-fold increase (p < 0.01) in mRNA levels for each, SOX9, type II collagen, and aggrecan during a 14-day culture period. Applying IHP in the presence of TGF-beta 3 further increased the mRNA levels for these proteins by 1.9-, 3.3-, and 1.6-fold, respectively, by day 14. Chondrogenic mRNA levels were increased with just exposure to IHP. Extracellular matrix deposition of type II collagen and aggrecan increased in the pellets as a function of treatment conditions and time of culture. This study demonstrated adjunctive effects of IHP on TGF-beta 3-induced chondrogenesis and suggests that mechanical loading can facilitate articular cartilage tissue engineering.

Biochemical and functional modulation of the cartilage collagen network by IGF1, TGFβ2 and FGF2

Examine effects of insulin-like growth factor 1 (IGF1), transforming growth factor beta2 (TGFbeta2) and fibroblast growth factor 2 (FGF2) on proteoglycan and collagen network and biomechanical properties of the newly formed cartilage matrix. Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without FGF2, TGFbeta2 or IGF1 in the presence of 10% FCS. Proteoglycan content, collagen content, hydroxylysylpyridinoline cross-links and overall matrix metalloproteinase (MMP) activity in the culture medium were measured. Alginate disks cultured for 5 weeks were used to evaluate the effect of growth factors on mechanical properties of the construct by determining the equilibrium aggregate modulus and secant modulus. IGF1 increased collagen and proteoglycan deposition. FGF2 mainly decreased collagen deposition and TGFbeta2 proteoglycan deposition. A decrease in cross-links was observed in matrix produced by chondrocytes cultured in the presence of TGFbeta2. IGF1 and FGF2 had no influence on the number of cross-links per collagen molecule. Overall MMP activity was significantly higher in culture medium of cells cultured with FGF2. TGFbeta2 and IGF1 had no effect on MMP activity. After 35 days of culture, the matrix produced under influence of IGF1 had a lower permeability and a trend to increase stiffness. FGF2 showed a trend to lower both properties. TGFbeta2 had no effect on these parameters. IGF1, TGFbeta2 and FGF2 had differential effects on collagen network formation. Of the three growth factors tested, IGF1 seems to be best in promoting the formation of a functional collagen network since it increased proteoglycan and collagen deposition and improved the mechanical properties.

Role of the Proteasome in TGF-β Signaling in Lens Epithelial Cells

The durability of the ubiquitin proteasome pathway in the mammalian lens makes this enzyme system a potential contributor to certain cataracts and posterior capsular opacification (PCO). The present study addresses proteasome involvement in TGF-beta induced, cataract-associated gene activation in human lens cells. HLE B-3 cells were treated with TGF-beta, in combination with the proteasome inhibitors MG-132 or lactacystin. TGF-beta target gene expression was measured by semiquantitative RT-PCR. Annexin-FITC staining and flow cytometry were used to assess apoptosis levels. Western blot analyses were performed with anti-SnoN and anti-Smad2 antibodies. TGF-beta induced the expression of alpha-smooth muscle actin, fibronectin, and TGF-beta-inducible gene mRNA in HLE B-3 cells and primary cultured human lens cells from donor tissues. TGF-beta also induced a time-dependent decrease in the level of the Smad repressor SnoN. Gamma-glutamyl-cysteine synthetase (gamma-GCS) mRNA levels decreased in the presence of TGF-beta. Proteasome inhibitor cotreatment blocked the induction of alpha-SMA mRNA, the loss of SnoN protein, the decrease in gamma-GCS mRNA, and TGF-beta-induced apoptosis. The HLE B-3 cell line and primary cultured human lens cells respond similarly to TGF-beta treatments by activating cataract-related gene expression. This response in both of these model systems is blocked by inhibiting the proteasome. This suggests that the proteasome can mediate cataract and PCO-associated changes and therefore is a novel target of medical therapy.

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor beta1 (TGF-beta1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-beta1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.

Chondrogenic differentiation of murine and human mesenchymal stromal cells in a hyaluronic acid scaffold: Differences in gene expression and cell morphology

Chondrogenesis is a complex process characterized by a sequence of different steps that start with the condensation of the cells, followed by the expression of specific components, such as collagens and proteoglycans. We evaluated in vitro chondrogenic differentiation of C3H10T1/2 murine mesenchymal cells and compared them with human mesenchymal stromal cells (h-MSCs) in a hyaluronic acid scaffold. We analyzed (from day 0 to day 28) cellular morphology, proliferation, and chondrogenic/osteogenic gene expression at different time points. Our data demonstrate that, during chondrogenic differentiation, murine cells proliferate both in the absence and presence of TGFbeta, while h-MSCs require the presence of this activating factor. Murine cells, even if viable, differentiate on hyaluronan scaffold, maintain a fibroblastic morphology, and form a capsule outside the scaffold. At the mRNA level, murine cells showed a decrease in collagen type I combined with a significant increase in collagen type II (from day 0), and aggrecan (on day 28), as found for h-MSCs. Immunohistochemical data confirmed that chondrogenic differentiation of murine cells, induced by TGFbeta, occurred only in some restricted areas inside the scaffold that were positive to collagen type II, but did not show a cartilage-like tissue structure, as we had found using h-MSCs. These data demonstrate that C3H10T1/2 murine cells, widely used as a chondrogenic model, show a different sequence of chondrogenic events in hyaluronic acid scaffold, compared with primary h-MSCs.

ERK1/2 and p38 MAP kinase control MMP‐2, MT1‐MMP, and TIMP action and affect cell migration: A comparison between mesothelioma and mesothelial cells

Pleural malignant mesothelioma is a locally aggressive tumor of mesothelial cell origin. In other tumor types high expression of matrix metalloproteinase (MMP)-2, together with membrane-type1-MMP (MT1-MMP), and low levels of the tissue inhibitor of MMP (TIMP)-2 have been correlated with aggressive tumor progression and low survival rates. Therefore, we compared the expression and activation of these three factors and their regulation by two mesothelioma associated growth factors, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)-beta1 in six human mesothelioma and one mesothelial cell line. Polymerase chain reaction (PCR), immunoblotting, zymography, and small inhibitory RNAs (siRNA) were used to study gene expression, protein activation, and signal transduction. To proof the relevance of our in vitro data immunohistochemistry was performed in tissue sections. PDGF-BB induced, while TGF-beta1 inhibited cell proliferation. PDGF-BB was a chemoattractant for mesothelial cells, and its effect was increased in the presence of TGF-beta1. TGF-beta1 stimulated the de novo synthesis of pro-MMP-2 in both cell types. Pro-MMP-2 synthesis involved p38 MAP kinase. In cell culture and tissue sections only mesothelial cells expressed MT1-MMP. Migration of mesothelioma cells was dependent on the presence of MT1-MMP. Migration, but not proliferation of mesothelioma cells was inhibited by oleoyl-N-hydroxylamide, TIMP-2, and siRNA for MT1-MMP. Our data suggest that in mesothelioma cells the phosphorylation of p38 MAP kinase is deregulated and is involved in pro-MMP-2 expression. Mesothelioma progression depends on an interaction with mesothelial cells that provide MT1-MMP necessary to activate pro-MMP-2 to facilitate migration through an extracellular matrix (ECM) layer.

The proteome of mouse mucosal mast cell homologues: The role of transforming growth factor β1

Mast cells migrate to the mucosal epithelium during intestinal nematode infections in mice, where they express abundant mucosal mast cell-specific proteases, mouse mast cell protease-1 and -2 (MCPT1 and MCPT2). Expression of these proteases is strictly controlled by transforming growth factor-beta1 (TGF-beta1) in the epithelium. In vitro homologues of mucosal mast cells are generated by culturing bone marrow-derived mast cells (BMMC) in the presence of TGF-beta1. We examined the proteome of BMMC cultured either in the presence of TGF-beta1 (n = 5) or of a neutralising anti-TGF-beta1 antibody (n = 5). Cell extracts were examined by 2-DE, and changes in expression levels of protein spots were determined by densitometry. Spots of interest were identified by tryptic peptide mapping. In addition to the up-regulation of MCPT1 and MCPT2, which accounted for approximately 40% of all soluble protein in the TGF-beta1 treated cells, MCPT7 was modestly up-regulated by TGF-beta1, and calnexin was up-regulated fivefold. A 7.6-fold down-regulation of galectin-1 was verified by Western blotting and FACS analysis. Galectin-1 is located on the cell surface where it mediates cellular adhesion to basement membranes. Regulation of its expression by TGF-beta1 may be of relevance to mast cell adhesion within the epithelium.

Effect of transforming growth factor-β1 on human intrahepatic cholangiocarcinoma cell growth

To elucidate the biological effects of transforming growth factor-beta1 (TGF-beta1) on intrahepatic cholan-giocarcinoma (ICC). We investigated the effects of TGF-beta1 on human ICC cell lines (HuCCT1, MEC, and HuH-28) by monitoring the influence of TGF-beta1 on tumor growth and interleukin-6 (IL-6) expression in ICC cells. All three human ICC cell lines produced TGF-beta1 and demonstrated accelerated growth in the presence of TGF-beta1 with no apoptotic effect. Studies on HuCCT1 revealed a TGF-beta1-induced stimulation of the expression of TGF-beta1, as well as a decrease in TGF-beta1 mRNA expression induced by neutralizing anti-TGF-beta1 antibody. These results indicate that TGF-beta1 stimulates the production and function of TGF-beta1 in an autocrine fashion. Further, IL-6 secretion was observed in all three cell lines and exhibited an inhibitory response to neutralizing anti-TGF-beta1 antibody. Experiments using HuCCT1 revealed a TGF-beta1-induced acceleration of IL-6 protein expression and mRNA levels. These findings demonstrate a functional interaction between TGF-beta1 and IL-6. All three cell lines proliferated in the presence of IL-6. In contrast, TGF-beta1 induced no growth effect in HuCCT1 in the presence of small interfering RNA against a specific cell surface receptor of IL-6 and signal transducer and activator of transcription-3. ICC cells produce TGF-beta1 and confer a TGF-beta1-induced growth effect in an autocrine fashion. TGF-beta1 activates IL-6 production, and the functional interaction between TGF-beta1 and IL-6 contributes to ICC cell growth by TGF-beta1.