TGF-beta Type Research Articles

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.

Antifibrogenic effects of tamoxifen in a rat model of periportal hepatic fibrosis

It has been reported that tamoxifen may affect hepatoma cell growth in vitro by suppressing transforming growth factor beta-1 (TGF-beta1) expression, suggesting that tamoxifen might also retard fibrogenesis. Thus, we examined whether tamoxifen might suppress TGF-beta1 expression and consequently inhibit the process of hepatic fibrosis in vivo. To induce periportal hepatic fibrosis, 50 male adult Sprague-Dawley rats were injected with 0.62 mmol/kg of allyl alcohol, intraperitoneally, twice a week for 8 weeks. Hepatic fibrosis scores, intrahepatic collagen levels and plasma TGF-beta1 expression levels were evaluated in three groups of 10 rats orally administered tamoxifen at 1, 5 and 10 mg/kg, respectively, and in 20 controls. Messenger RNAs (mRNAs) encoding TGF-beta1 and TGF-beta receptors in liver tissue were semiquantified using reverse transcriptase polymerase chain reaction. Hepatic fibrosis scores decreased progressively as the dose of tamoxifen increased, resulting in a significant change in rats treated with tamoxifen at 10 mg/kg compared with controls (P=0.018). Intrahepatic collagen content was significantly less in the group treated with tamoxifen at 10 mg/kg compared with the control (P=0.045). Plasma TGF-beta1 levels were also significantly lower in rats treated with tamoxifen at 10 mg/kg compared with controls (P=0.007). All three concentrations of tamoxifen tested decreased the expression levels of hepatic TGF-beta1 mRNA and type I TGF-beta receptor (TGF-beta RI) mRNA to similar extents. Tamoxifen seems to inhibit the process of hepatic fibrosis dose-dependently by suppressing the transcription of TGF-beta1 and TGF-beta RI in an experimental model of periportal hepatic fibrosis.

Expression and Cyclic Change of Betaglycan in the Rat Oviduct

Although the fallopian tube provides a sufficient environment for fertilization and early embryo development, the mechanism by which it does this is unclear. It is known that the transforming growth factor beta (TGFbeta) superfamily plays important roles in various reproductive functions. Betaglycan, originally characterized as a TGF beta type III receptor lacking a clearly identifiable signaling motif, has been shown to be important for the high-affinity binding of TGF betas to the type II receptor. To our knowledge, there has been no study showing expression of betaglycan in the rat oviduct. Therefore, in this study, we examined the distribution of betaglycan in various rat tissues and its expression patterns in the oviduct during the estrous cycle. Northern blot analysis of various rat tissues showed that the adrenal gland, ovary and oviduct contained abundant amounts of 6.4-kb betaglycan mRNA. Furthermore, the mRNA level of betaglycan was highest after the LH surge that induced ovulation. The betaglycan protein, detected using immunohistochemistry, was especially abundant in the epithelium of the oviduct. Furthermore, in pregnant mare serum gonadotropin (PMSG) primed-rats, the expression of betaglycan was increased significantly by stimulation with human chorionic gonadotropin (hCG). RT-PCR analysis showed co-localization of other TGF beta family receptors (TGF beta types I and II, activin receptor types Ia and Ib and activin receptor types IIa and IIb) with betaglycan in the oviduct. Since betaglycan along with other TGF beta family receptors is abundantly expressed in the epithelium of the oviduct and its expression changes during estrous, it may also play an important role in the oviduct.

TGF-.BETA. Inhibits Vascular Sprouting through TGF-.BETA. type I Receptor in the Mouse Embryonic Aorta

Organogenesis accompanies the establishment of the vascular system which begins with sprouting angiogenesis. Vascular endothelial growth factor (VEGF) provides the primary stimulation in the vascular sprouting process but the negative regulation of this process remains unclear. This study examined the role of the transforming growth factor-beta (TGF-beta) superfamily in vascular sprouting using a three-dimensional dorsal aorta culture system, in which the dissected tissue was embedded in type I collagen gel. The cultures were maintained under hypoxic conditions to enhance the expression of Flk-1, a receptor for VEGF, thereby ensuring the responsibility to VEGF. Under the culture conditions employed, the dorsal aorta formed many cord-like structures in response to VEGF. To examine the role of TGF-beta in vascular sprouting, each member of the TGF-beta superfamily was applied to this culture system. TGF-beta1, as well as TGF-beta2 and TGF-beta 3, inhibited capillary formation. Likewise, activin A, another member of TGF-beta superfamily, also abolished vascular sprouting, but bone morphogenetic protein 2 did not noticeably change the morphology. Both neutralizing anti-TGF-beta1 antibody and TGF-beta type I receptor (ALK5) inhibitor partially reversed the inhibitory effect of TGF-beta1. Furthermore, down-regulation of ALK5 with small interfering RNA rather than activin receptor-like kinase-1 (ALK1) reversed the effect of TGF-beta1. These data suggest that TGF-beta superfamily may act as an inhibitor of vascular sprouting mainly through ALK5 signaling pathway. We propose that VEGF may antagonize the TGF-beta autoregulatory action to initiate vascular sprouting.

Human T-Cell Leukemia Virus Type 1 Tax Activates Caveolin-1 Gene Expression

Caveolae are plasma membrane invaginations that function as regulators of signal transduction. Caveolins are a class of oligomeric structural proteins that are both necessary and sufficient for caveolae formation. Caveolin-1 has been shown to regulate multiple cancer-associated processes including cellular transformation, tumor growth, cell migration and metastasis, cell death and survival, multidrug resistance, and angiogenesis. Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). We examined expression of caveolin-1 and caveolin-2 in HTLV-1-infected T-cell lines and primary ATL cells. These cells expressed high levels of caveolin-1 mRNA compared with uninfected T-cell lines and normal PBMCs. However, caveolin-2 mRNA expression was not restricted to HTLV-1-infected T cells. Immunohistochemical staining of caveolin-1 in ATL lymph nodes and skin lesions showed that primary ATL cells were indeed stained strongly positive for caveolin-1. In vitro infection of a human T-cell line with HTLV-1 induced caveolin-1 mRNA expression. The viral protein Tax transcriptionally activated caveolin-1 gene. Using Tax mutants, we demonstrated that Tax-induced caveolin-1 expression required the NF-kappaB and CREB signaling pathways. TGF-beta is an inhibitor of T-cell proliferation and cytotoxicity. HTLV-1-infected T-cell lines and ATL cells are known to be resistant to TGF-beta-induced growth inhibition. We demonstrated that caveolin-1 is able to suppress TGF-beta signaling at the transcriptional level in T cells. Caveolin-1 colocalized with TGF-beta type I receptor in an HTLV-1-infected T-cell line. Thus, we describe a new function of Tax, as a repressor of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

Generation of mice with conditionally activated transforming growth factor beta signaling through the TβRI/ALK5 receptor

We generated a transgenic mouse strain (LSL-TbetaRI(CA)) containing a latent constitutively active TGFbeta type I receptor (TbetaRI/ALK5) by using a knock-in strategy into the X chromosome-linked hypoxanthine phosphoribosyl-transferase (Hprt) locus. Transgene expression, under the control of the ubiquitous CAG (human cytomegalovirus enhancer and chicken beta-actin) promoter, is repressed by a floxed transcriptional "Stop" (LSL, Lox-Stop-Lox). In the presence of cre-recombinase, the "Stop" is excised to allow TbetaRI(CA) transgene expression. We showed that restricted expression of TbetaRI(CA) in T lymphocytes efficiently activates TGFbeta signaling and rescues the T-cell autoimmune disorders of TGFbetaRII conditional knockouts. Unexpectedly, our study reveals that TGFbeta signaling upregulation controls T-cell activation but does not impair their development or their peripheral homeostasis. In addition to the information provided on TGFbeta effects on T-cell biology, LSL-TbetaRI(CA) mouse constitutes an attractive tool to address the effect of TGFbeta signaling upregulation in any cell type expressing the cre-recombinase.

Effect of inhaled budesonide on surfactant protein expression in asthmatic mice

Pulmonary surfactant dysfunction may significantly contribute to small airway obstruction during the asthmatic response. Inhaled corticosteroids have been routinely used in the treatment of asthma, but neither its exact role nor its regulation on pulmonary surfactant is completely understood. The aim of this study was to determine the effect of budesonide on surfactant protein (SP) expression in asthmatic mice. Moreover, we investigated the function of transforming growth factor (TGF) beta signaling in the pulmonary surfactant system to identify a novel target for the treatment of asthma. Mice were sensitized and challenged by ovalbumin (OVA) to establish a murine model of asthma. To assess the effect of budesonide on asthmatic mice, animals were treated with aerosolized budesonide before OVA challenge. The levels of TGF-beta(1) in bronchoalveolar lavage fluid were analyzed by ELISA. The expressions of TGF-beta type I receptor, TGF-beta type II receptor, Smad2/3, Smad4, Smad7, and SPs were measured by immunochemistry technique and computerized image analysis system. SP-A and SP-B were significantly decreased after allergic sensitization and challenge, accompanied by active TGF-beta/Smad signal transduction. A dramatic increase in the expressions of SP-A and SP-B was observed after budesonide treatment. This was also accompanied by up-regulation of Smad7 expression and down-regulation of TGF-beta type I receptor expression. A possible explanation for the result is that an early budesonide inhaled treatment inhibits TGF-beta-induced reduction of SP-A and SP-B expression through inhibition of active TGF-beta/Smad signal transduction pathway in asthmatic mice. TGF-beta signaling may be a potentially important therapeutic target for antiasthma drugs.

Identification of novel Smad2 and Smad3 associated proteins in response to TGF-beta1.

Transforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-beta1 signals through the TGF-beta type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A non-transformed human cell line was transduced with Myc-His(6)-Smad2 or Myc-His(6)-Smad3-expressing retrovirus and was treated with TGF-beta1. Myc-His(6)-Smad2 or Myc-His(6)-Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-beta1 signaling.

Prostate tumor progression is mediated by a paracrine TGF-β/Wnt3a signaling axis

Transforming growth factor (TGF)-beta is an important paracrine factor in tumorigenesis. Ligand binding of the type I and II TGF-beta receptors initiate downstream signaling. The role of stromal TGF-beta signaling in prostate cancer progression is unknown. In mice, the conditional stromal knockout of the TGF-beta type II receptor expression (Tgfbr2(fspKO)) resulted in the development of prostatic intraepithelial neoplasia and progression to adenocarcinoma within 7 months. Clinically, we observed a loss of TGF-beta receptor type II expression in 69% of human prostate cancer-associated stroma, compared to 15% of stroma associated with benign tissues (n=140, P-value <0.0001). To investigate the mechanism of paracrine TGF-beta signaling in prostate cancer progression, we compared the effect of the prostatic stromal cells from Tgfbr2(fspKO) and floxed TGF-beta type II receptor Tgfbr2(floxE2/floxE2) mice on LNCaP human prostate cancer cells in vitro and tissue recombination xenografts. Induction of LNCaP cell proliferation and tumorigenesis was observed by Tgfbr2(fspKO) prostate stroma as a result of elevated Wnt3a expression. Neutralizing antibodies to Wnt3a reversed LNCaP tumorigenesis. The TGF-beta inhibition of Wnt3a expression was in part through the suppression of Stat3 activity on the Wnt3a promoter. In conclusion, the frequent loss of stromal TGF-beta type II receptor expression in human prostate cancer can relieve the paracrine suppression of Wnt3a expression.

Adoptive Transfer of Tumor-Reactive Transforming Growth Factor-β–Insensitive Cytolytic T Cells for Treatment of Established Mouse Renca Tumors

To test, using a mouse renal cancer, Renca, whether adoptive transfer of tumor-reactive transforming growth factor (TGF)-beta-insensitive cytolytic T cells can inhibit tumor progression. Cytolytic T cells were isolated from the spleen of male Balb/c mice repeatedly primed with irradiated Renca cells. They were expanded ex vivo and were rendered TGF-beta-insensitive by infecting with a retrovirus containing dominant negative TGF-beta type II receptor. These tumor reactive TGF-beta-insensitive cytolytic T cells showed a specific and robust tumor killing activity against Renca cells, but not irrelevant cells, using an in vitro cytotoxic assay. Adoptive transfer of cytolytic T cells was performed in mice 10 days after they were challenged with Renca cells (5 x 10(5)) by tail vein injection. At 30 days after the adoptive transfer, the pulmonary tumor counts in mice who had received TGF-beta-insensitive cytolytic T cells (mean +/- standard deviation 130 +/- 140) was significantly less than those in mice that had received TGF-beta-sensitive cytolytic T cells (305 +/- 60) or in mice had received naive cytolytic T cells (375 +/- 50; P < .01). Kaplan-Meier survival analysis indicated that mice that had received adoptive transfer of TGF-beta-insensitive cytolytic T cells had a significantly greater rate of survival (75%) compared with mice that had received TGF-beta-sensitive cytolytic T cells (35%) or naive cytolytic T cells (15%), respectively (P < .05). These results suggest that adoptive transfer of tumor-reactive TGF-beta-insensitive cytolytic T cells can warrant consideration for renal cell cancer immunotherapy.

Smad7 induces hepatic metastasis in colorectal cancer

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-β-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-β type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-β/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

MMP-13 is over-expressed in renal cell carcinoma bone metastasis and is induced by TGF-β1

Bone metastasis occurs frequently in renal cell carcinoma (RCC) patients causing significant morbidity by stimulating excessive osteolysis, yet the mechanisms responsible have been little studied. Matrix metalloproteinases (MMPs) are over-expressed in many cancer types and are believed to play a role in bone metastasis, however, the expression of MMPs in RCC bone metastasis (RBM) has not been investigated. Due to their ability to degrade the main component of organic bone matrix, type I collagen, we investigated the expression of MMP-1, -2, -8, -9, and -13 in RBM. By quantitative (q)RT-PCR, expression of MMP-13 was significantly increased in RBM tissues relative to that in RCC and adjacent normal kidney while no differences in the expression of MMP-1, -2, -8, or -9 mRNA were observed. Correspondingly, increased expression of MMP-13 protein was also observed in RBM relative to RCC by immunohistochemical analysis. Intriguingly, the expression of MMP-13 in the human RBM cell line RBM1-IT4 was stimulated by TGF-beta1, a growth factor abundant in the bone microenvironment and known to promote RBM-induced osteolysis in animals. Exposure of RBM1-IT4 cells to TGF-beta1 increased MMP-13 mRNA levels as well as the latent and active forms of MMP-13 protein. Further, stable expression of a dominant-negative TGF-beta type II receptor in RBM1-IT4 cells inhibited MMP-13 expression following TGF-beta1 exposure. These data suggest that MMP-13 expression is elevated in RBM relative to primary RCC and adjacent normal kidney, and is regulated at the cellular level by TGF-beta1.

Germline allele-specific expression of TGFBR1 confers an increased risk of colorectal cancer.

Much of the genetic predisposition to colorectal cancer (CRC) in humans is unexplained. Studying a Caucasian-dominated population in the United States, we showed that germline allele-specific expression (ASE) of the gene encoding transforming growth factor-beta (TGF-beta) type I receptor, TGFBR1, is a quantitative trait that occurs in 10 to 20% of CRC patients and 1 to 3% of controls. ASE results in reduced expression of the gene, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases. Although subtle, the reduction in constitutive TGFBR1 expression alters SMAD-mediated TGF-beta signaling. Two major TGFBR1 haplotypes are predominant among ASE cases, which suggests ancestral mutations, but causative germline changes have not been identified. Conservative estimates suggest that ASE confers a substantially increased risk of CRC (odds ratio, 8.7; 95% confidence interval, 2.6 to 29.1), but these estimates require confirmation and will probably show ethnic differences.

Yeast-secreted Recombinant Extracellular Domain of Human CD105 Antigen is Able to Bind TGF-beta Type II Receptor In Vitro

Human CD105 antigen, a type I integral membrane glycoprotein, is expressed as homodimer and oligomer by human endothelial cells, and forms a heteromeric association with TGF-beta signaling receptors I and II. Several mutations of CD105 antigen gene are involved in a vascular disorder known as hereditary hemorrhagic telangiectasia type 1. The proposed mechanism by which CD105 is involved in said disorder is haploinsufficiency. We report expression and characterization of human CD105 antigen extracellular domain in yeast Saccharomyces cerevisiae. Different strategies to influence the release of heterologous proteins in the medium, such as alteration of cell wall integrity or coexpression of protein disulfide isomerase, were addressed. Purified extracellular domain of human CD105 antigen retains capacity to bind human TGF-beta receptor II in vitro.

Recognition and characterization of TGF-β receptor interacting protein 1 (TRIP-1) containing WD40 repeats from Clonorchis sinensis by bioinformatics, cloning, and expression in Escherichia coli

A cDNA clone encoding a homologue of transforming growth factor beta (TGF-beta) receptor interacting protein 1 (TRIP-1) was recognized and isolated from full-length cDNA plasmid library of Clonorchis sinensis adult. TRIP-1 is a bifunctional molecule in all eukaryote, which modulates the signaling pathway of TGF-beta as a phosphorylation substrate of TGF-beta type II receptor kinase and controls ribosome assembly and mRNA translation as p36 subunit of the eukaryotic translation initiation factor 3. The structural and immunological characteristics of TRIP-1 from C. sinensis (CsTRIP-1) were analyzed by bioinformatics. The complete coding sequence was expressed in Escherichia coli, and the purified recombinant product was obtained. Western blotting with mixed sera from clonorchiasis patients was positive, whereas the normal was negative, suggesting it is a candidate of diagnostic antigen for clonorchiasis. CsTRIP-1 will aid to explore interaction between host and the parasite as well as the mechanism by which TGF-beta controls the development of C. sinensis and participates in the pathogenesis.

Expression and function of alternative splice variants of the mouse TGF‐β type I receptor

Mouse TGF-beta type I receptor, Alk5, encodes two types of mRNA generated by alternative splicing of four amino acids in the extracellular region. In the present study, the expression and function of alternative splice variants of Alk5, i.e., Alk5-L (Alk5 with the four amino acids) and Alk5-S (Alk5 without the four amino acids), were examined. Expression of Alk5 was detected in all examined tissues, and no significant differences in the ratio of Alk5-S to Alk5-L were detected between tissues. Expression of Alk5 was also detected in various cells, with Alk5-L being especially abundant in A-6 ES cells when it was examined by RT-PCR and subsequent image analyses. No clear difference in Smad2 phosphorylation in response to treatment with TGF-beta(1) was detected between L17 cells expressing Alk5-S and those expressing Alk5-L. Consistent with these results, transcription mediated by Alk5-S in L17 cells treated with TGF-beta(1) was comparable to that mediated by Alk5-L. In addition, alternative splicing had no effect on transcriptional activation induced by GDF-8, a member of the TGF-beta family. The present results indicate ubiquitous expression of Alk5 isoforms in mouse tissues and cells, and insignificant effects of alternative splicing on signaling induced by TGF-beta(1) and GDF-8.

173 Autologous Bone Marrow-Derived Cells for Accelerating the Healing of Human Chronic Wounds

We have previously shown that resident fibroblasts from human chronic wounds are phenotypically abnormal, displaying an impaired response to TGF-beta 1, decreased TGF-beta Type II receptor expression, and decreased phosphorylation of Smad2, Smad3, and p42/44 MAPK. We now report that these human cells delay healing by approximately 30% when implanted in mouse incisional wounds and in a dorsal flap model. These new findings point to the need for the transplantation of new healthy cells in human chronic wounds. Bioengineered living skin constructs can deliver new cells to chronic wounds, at least for a limited period of time. However, most available bioengineered skin constructs consist of already differentiated allogeneic cells, which do not allow for the possibility of engraftment and reconstitution of wound structures. Recently, in a preliminary report, we have shown that autologous bone marrow-derived cells can dramatically enhance the healing of recalcitrant chronic wounds when all other treatments have failed. Our present efforts are now focused on characterizing the bone marrow cells involved in this stimulation of wound healing and in developing better ways for their topical delivery to the wound. Here we present evidence that a fibrin construct may be an ideal way of applying bone marrow-derived cells to human chronic wounds. Autologous bone marrow cells are allowed to grow in culture under conditions favoring the survival and proliferation of mesenchymal stem cells (MSC), as shown by surface markers and functional studies. This usually requires two weeks after bone marrow harvesting. The cellular component is then placed in a fibrinogen solution which, when simultaneously combined with thrombin through a CO2 driven flow, delivers a fine cell-containing fibrin polymer film to the wound. In vitro and in vivo experiments, both in mouse models and in human wounds, show the feasibility of this approach.

Fibroproliferation in LPS-induced airway remodeling and bleomycin-induced fibrosis share common patterns of gene expression

Chronic LPS inhalation causes submucosal thickening and airway narrowing. To address the hypothesis that environmental airway disease is, in part, a fibroproliferative lung disease, we exposed C57BL/6 mice daily to LPS by inhalation for up to 2 months followed by 1 month of recovery. C57BL/6 mice exposed to daily inhaled LPS had significantly enhanced mRNA expression of TGF-beta1, TIMP-1, fibronectin-1, and pro-collagen types I, III, and IV and show prominent submucosal expression of the myofibroblast markers desmin and alpha-smooth muscle actin. To further characterize global gene expression in airway fibroproliferation, we performed microarray analysis on total lung RNA from mice exposed to LPS both acutely and chronically. This analysis revealed a subset of genes typically associated with lung injury and repair, and ECM homeostasis. To further identify candidate genes specifically involved in generic fibroproliferation, we interrogated this analysis with genes induced in C57BL/6 mouse lung by bleomycin. This analysis yielded a list of 212 genes in common suggesting that there is a common subset of genes that regulate fibroproliferation in the lung independent of etiologic agent and site of injury.

Regulation of CCN2 mRNA expression and promoter activity in activated hepatic stellate cells

The matricellular protein connective tissue growth factor (CCN2) is considered a faithful marker of fibroblast activation in wound healing and in fibrosis. CCN2 is induced during activation of hepatic stellate cells (HSC). Here, we investigate the molecular basis of CCN2 gene expression in HSC. Fluoroscence activated cell sorting was used to investigate CCN2 expression in HSC in vivo in mice treated with CCl(4). CCN2 and TGF-beta mRNA expression were assessed by polymerase chain reaction as a function of culture-induced activation of HSC. CCN2 promoter/reporter constructs were used to map cis-acting elements required for basal and TGFbeta-induced CCN2 promoter activity. Real-time polymerase chain reaction analysis was used to further clarify signaling pathways required for CCN2 expression in HSC. CCl(4) administration in vivo increased CCN2 production by HSC. In vitro, expression of CCN2 and TGF-beta mRNA were concommitantly increased in mouse HSC between days 0 and 14 of culture. TGFbeta-induced CCN2 promoter activity required the Smad and Ets-1 elements in the CCN2 promoter and was reduced by TGFbeta type I receptor (ALK4/5/7) inhibition. CCN2 overexpression in activated HSC was ALK4/5/7-dependent. As CCN2 overexpression is a faithful marker of fibrogenesis, our data are consistent with the notion that signaling through TGFbeta type I receptors such as ALK5 contributes to the activation of HSC and hence ALK4/5/7 inhibition would be expected to be an appropriate treatment for liver fibrosis.

Antitumor Activity of EBV-specific T Lymphocytes Transduced With a Dominant Negative TGF-β Receptor

Transforming growth factor (TGF)-beta is produced in most human tumors and markedly inhibits tumor antigen-specific cellular immunity, representing a major obstacle to the success of tumor immunotherapy. TGF-beta is produced in Epstein-Barr virus (EBV)-positive Hodgkin disease and non-Hodgkin lymphoma both by the tumor cells and by infiltrating T-regulatory cells and may contribute the escape of these tumors from infused EBV-specific T cells. To determine whether tumor antigen-specific cytotoxic T lymphocytes (CTLs) can be shielded from the inhibitory effects of tumor-derived TGF-beta, we previously used a hemagglutinin-tagged dominant negative TGF-betaRII expressed from a retrovirus vector to provide CTLs with resistance to the inhibitory effects of TGF-beta in vitro. We now show that human tumor antigen-specific CTLs can be engineered to resist the inhibitory effects of tumor-derived TGF-beta both in vitro and in vivo using a clinical grade retrovirus vector in which the dominant negative TGF-beta type II receptor (DNRII) was modified to remove the immunogenic hemagglutinin tag. TGF-beta-resistant CTL had a functional advantage over unmodified CTL in the presence of TGF-beta-secreting EBV-positive lymphoma, and had enhanced antitumor activity, supporting the potential value of this countermeasure.