Induced Smad2 Phosphorylation Research Articles

Assembly of fibrillin microfibrils governs extracellular deposition of latent TGFβ

Control of the bioavailability of the growth factor TGFbeta is essential for tissue formation and homeostasis, yet precisely how latent TGFbeta is incorporated into the extracellular matrix is unknown. Here, we show that deposition of a large latent TGFbeta complex (LLC), which contains latent TGFbeta-binding protein 1 (LTBP-1), is directly dependent on the pericellular assembly of fibrillin microfibrils, which interact with fibronectin during higher-order fibrillogenesis. LTBP-1 formed pericellular arrays that colocalized with microfibrils, whereas fibrillin knockdown inhibited fibrillar LTBP-1 and/or LLC deposition. Blocking alpha5beta1 integrin or supplementing cultures with heparin, which both inhibited microfibril assembly, disrupted LTBP-1 deposition and enhanced Smad2 phosphorylation. Full-length LTBP-1 bound only weakly to N-terminal pro-fibrillin-1, but this association was strongly enhanced by heparin. The microfibril-associated glycoprotein MAGP-1 (MFAP-2) inhibited LTBP-1 binding to fibrillin-1 and stimulated Smad2 phosphorylation. By contrast, fibulin-4, which interacted strongly with full-length LTBP-1, did not induce Smad2 phosphorylation. Thus, LTBP-1 and/or LLC deposition is dependent on pericellular microfibril assembly and is governed by complex interactions between LTBP-1, heparan sulfate, fibrillin-1 and microfibril-associated molecules. In this way, microfibrils control TGFbeta bioavailability.

Smad2 Protects against TGF-β/Smad3-Mediated Renal Fibrosis

Smad2 and Smad3 interact and mediate TGF-beta signaling. Although Smad3 promotes fibrosis, the role of Smad2 in fibrogenesis is largely unknown. In this study, conditional deletion of Smad2 from the kidney tubular epithelial cells markedly enhanced fibrosis in response to unilateral ureteral obstruction. In vitro, Smad2 knockdown in tubular epithelial cells increased expression of collagen I, collagen III, and TIMP-1 and decreased expression of the matrix-degrading enzyme MMP-2 in response to TGF-beta1 compared with similarly treated wild-type cells. We obtained similar results in Smad2-knockout fibroblasts. Mechanistically, Smad2 deletion promoted fibrosis through enhanced TGF-beta/Smad3 signaling, evidenced by greater Smad3 phosphorylation, nuclear translocation, promoter activity, and binding of Smad3 to a collagen promoter (COL1A2). Moreover, deletion of Smad2 increased autoinduction of TGF-beta1. Conversely, overexpression of Smad2 attenuated TGF-beta1-induced Smad3 phosphorylation and collagen I matrix expression in tubular epithelial cells. In conclusion, in contrast to Smad3, Smad2 protects against TGF-beta-mediated fibrosis by counteracting TGF-beta/Smad3 signaling.

Radiotherapy Suppresses Angiogenesis in Mice through TGF-βRI/ALK5-Dependent Inhibition of Endothelial Cell Sprouting

BackgroundRadiotherapy is widely used to treat cancer. While rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence indicates that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity. In spite of its clinical relevance, such putative anti-angiogenic effect of radiotherapy has not been thoroughly characterized. We have investigated the effect of ionizing radiation on angiogenesis using in vivo, ex vivo and in vitro experimental models in combination with genetic and pharmacological interventions.Principal FindingsHere we show that high doses ionizing radiation locally suppressed VEGF- and FGF-2-induced Matrigel plug angiogenesis in mice in vivo and prevented endothelial cell sprouting from mouse aortic rings following in vivo or ex vivo irradiation. Quiescent human endothelial cells exposed to ionizing radiation in vitro resisted apoptosis, demonstrated reduced sprouting, migration and proliferation capacities, showed enhanced adhesion to matrix proteins, and underwent premature senescence. Irradiation induced the expression of P53 and P21 proteins in endothelial cells, but p53 or p21 deficiency and P21 silencing did not prevent radiation-induced inhibition of sprouting or proliferation. Radiation induced Smad-2 phosphorylation in skin in vivo and in endothelial cells in vitro. Inhibition of the TGF-β type I receptor ALK5 rescued deficient endothelial cell sprouting and migration but not proliferation in vitro and restored defective Matrigel plug angiogenesis in irradiated mice in vivo. ALK5 inhibition, however, did not rescue deficient proliferation. Notch signaling, known to hinder angiogenesis, was activated by radiation but its inhibition, alone or in combination with ALK5 inhibition, did not rescue suppressed proliferation.ConclusionsThese results demonstrate that irradiation of quiescent endothelial cells suppresses subsequent angiogenesis and that ALK5 is a critical mediator of this suppression. These results extend our understanding of radiotherapy-induced endothelial dysfunctions, relevant to both therapeutic and unwanted effects of radiotherapy.

220 Targeting CEA Rescues Proliferation Inhibiting Effects of TGF-β Signaling Pathway in Colorectal Cancers

Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGFβ signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGFβ response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases.

224 Specific Nucleotides That Flank Coding Microsatellites of Activin Receptor 2 (ACVR2) Determine Exonic Selectivity and Control Frameshift Mutation Rates in Defective DNA Mismatch Repair (MMR)

Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGFβ signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGFβ response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases.

Abstract 1491: CEA interacts with TGF-β receptor and inhibits TGF-β signaling in colorectal cancers

Abstract Carcinoembryonic antigen (CEA) is a tumor marker for the clinical management of colorectal cancer (CRC). The elevated blood levels of CEA are associated with metastasis and poor prognosis in CRC. There is mounting evidence that CEA enhances the metastatic potential of cancer cells. CEA increases the ability of weakly metastatic CRC to colonize the liver and to develop spontaneous hematogeneous liver and lung metastases. CEA expression has also been related with resistance to cytotoxic chemotherapy and to anoikis, a form of apoptosis caused by detachment from cell matrix. Yet the mechanism of CEA mediated metastasis is only partially understood. The TGF-β (transforming growth factor beta) signaling pathway contributes to tumorigenesis by controlling several biological processes, including cell proliferation, differentiation, migration and apoptosis. It has been reported that TGF-β regulates CEA transcription and secretion, however, little is known about the effects of CEA on TGF-β signaling. Aims: Based on the above facts, we focused on the influence of CEA on the TGF-β signaling in both normal cells and colorectal cancer cells. Results: Our preliminary data showed that CEA directly interacted with TGF-β receptors. Overexpression of CEA blocked TGF-β induced SMAD3 phosphorylation, SMAD3 translocation to nuclear and the downregulation of c-myc transcription. Targeting CEA with anti-CEA antibody rescued TGF-β response in CRC cell lines with elevated CEA expression, thereby restoring the inhibitory effects of TGF-β on the proliferation of these cancer cells. Finally, in animal experiment, we found that CEA enhanced survival of colorectal cancer cell in both local colonization and liver metastasis. Conclusion: Since CEA is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors, specific antibodies targeting CEA have been developed as a novel therapeutic approach for treatment of tumors expressing CEA on their surface. Based on our study, it may be helpful to combine CEA antibody and TGF-β to inhibit cancer cell proliferation and metastasis in some cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1491.

Curcumin Inhibits Transforming Growth Factor-β Activity via Inhibition of Smad Signaling in HK-2 Cells

Background: Curcumin, a polyphenolic compound derived from rhizomes of Curcuma spp., has been shown to possess potent anti-fibrotic properties. Here, we investigate the role of curcumin in modulating the profibrotic action of TGF-β in human proximal tubule cells (HK-2) and its underlying mechanisms. Methods: HK-2 cells were stimulated with 5 ng/ml TGF-β₁. The effects of curcumin on TGF-β₁-regulated gene expression and Smad phosphorylation were analyzed by RT-PCR, ELISA and Western blotting. Results:Curcumin inhibited TGF-β₁-induced plasminogen activator inhibitor-1 (PAI-1), α-smooth muscle actin (α-SMA) mRNA and protein expression. Curcumin suppressed not only TGF-β₁-induced Smad2 phosphorylation in a dose- and time-dependent manner, but also the nuclear accumulation of receptor-regulated Smads (R-Smad), Smad2 and Smad3. A serine/threonine protein phosphatase inhibitor (microcystin) could partly reverse the inhibitory effect of curcumin on Smad phosphorylation. Conclusions: Curcumin blocks the profibrotic actions of TGF-β on HK-2 cells through the down-regulation of the Smad signaling pathway, and curcumin may have some similar effect as serine/threonine protein phosphatases. Our findings suggest curcumin as a potential candidate for treatment of tubulointerstitial fibrosis.

Dual Nature of TGF-β1 in Osteoblastic Differentiation of Human Periodontal Ligament Cells

This study investigated the effect of various treatments with TGF-β1 in osteogenic differentiation of human periodontal ligament (HPDL) cells. HPDL cells were treated with single 1 ng/ml, 100 ng/ml or multiple (12-h intervals) 1 ng/ml TGF-β1. Alkaline phosphatase (ALP) activity was detected by ALP activity staining at 4 days. Phospho-Smad3 was detected by western blotting and transcription of osteogenic differentiation markers (RUNX2, ALPL, IBSP: bone sialoprotein, BGLAP: osteocalcin) were determined by real-time PCR at various culture periods. Cells were pretreated with or without SB505124, a TGF-β/Smad pathway inhibitor, for 1 h before 1 ng/ml TGF-β1 treatment at 12-h intervals. ALP activity and phosphorylated Smad3 were detected. Single treatment with 1 ng/ml TGF-β1 significantly induced Smad3 phosphorylation, subsequent ALP upregulation and transcription of osteogenic differentiation markers, compared to these inductions with 100 ng/ml TGF-β1 treatment. In multiple treatments with 1 ng/ml TGF-β1 at 12-h intervals, only the first treatment significantly induced Smad3 phosphorylation but not the succeeding treatments. Inductive effects of single 1 ng/ml TGF-β1 in osteogenic differentiation were almost completely abolished by multiple treatments. Furthermore, multiple treatments with 1 ng/ml TGF-β1 did not induce ALP activity although the TGF-β/Smad pathway was inhibited by SB505124. Single treatment with low dose TGF-β1 significantly induced osteogenic differentiation. Multiple treatments inhibited osteogenic differentiation despite inhibition of TGF-β/Smad pathway. These findings suggest that TGF-β1 both positively and negatively regulates osteogenic differentiation and the inhibitory effect may be mediated by the non-Smad signaling pathway in HPDL cells.

Abstract C136: An analysis of BCAR3-mediated regulation of p130Cas localization and focal adhesion formation in breast cancer cells

Abstract BCAR3 and p130Cas, two focal adhesion proteins that bind to one another and that were previously identified in a screen for genes that confer anti-estrogen resistance in ER+ breast cancer cells, were analyzed in breast cancer cell lines to determine the effect of BCAR3 expression on cell morphology and protein localization including the Src-mediated redistribution of p130Cas. The effect of altered BCAR3 expression and role of BCAR3 and p130Cas association was studied in MCF7 cells stably over-expressing wildtype BCAR3 or BCAR3 containing an R743A missense mutation that prevents p130Cas association. Protein localization and cellular morphology were examined by immunofluorescence and Western analyses of cells were conducted to observe p130Cas post-translational modifications and the potential involvement of the TGFβ and β-catenin signaling pathways. Stable over-expression of BCAR3 in MCF7 epithelial cells induces the loss of cohesive association between cells and the formation of membrane projections as well as large focal adhesions that contain abundant p130Cas. This BCAR3-mediated increase in p130Cas localization to focal adhesions is independent of the association of BCAR3 and p130Cas. Src, which is known to phosphorylate p130Cas, also induces augmented localization of p130Cas and this effect is also further enhanced by BCAR3 expression. Once again, physical interaction between BCAR3 and p130Cas is not necessary for the ability of BCAR3 to increase Src-mediated redistribution of p130Cas to focal adhesions. In spite of inducing a loss of epithelial morphology in MCF7 cells, BCAR3 over-expression did not induce SMAD2 phosphorylation or nuclear translocation of β-catenin, suggesting that neither the TGFβ or β-catenin signaling pathways are activated in response to BCAR3 over-expression in epithelial breast cancer cells. Consistent with these results, shRNA-mediated reduction of BCAR3 expression in MDA-231 mesenchymal cells ablates basal p130Cas phosphorylation, alters p130Cas and focal adhesion localization and induces an epithelial-like growth pattern with attenuated stress fiber formation and decreased tensin expression. The inhibition of stress fiber formation in MDA-231 cells prevents p130Cas phosphorylation. BCAR3 expression regulates the morphology and cytoskeleton of epithelial and mesenchymal breast cancer cells by affecting focal adhesion formation, p130Cas localization and the cytoskeletal response to Src signaling. The effect of BCAR3 appears not to involve either the TGFβ or β-catenin signaling pathways and is largely independent of BCAR3's direct physical interaction with p130Cas. Our current studies are seeking to examine the role of BCAR3's SH2 domain and serine-proline-rich region in the regulation of focal adhesion biology. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C136.

Differential TGF-β Signaling in Retinal Vascular Cells: A Role in Diabetic Retinopathy?

Purpose. An early hallmark of preclinical diabetic retinopathy is thickening of the capillary basal lamina (BL). TGF-beta, a multipotent cytokine acting through its receptors ALK5 and -1, has been postulated to be involved in this phenomenon. In light of this possible role, TGF-beta signaling and its downstream molecular effects were characterized in cultured vascular endothelial cells and pericytes of the retina. Methods. Bovine retinal endothelial cells and pericytes were stimulated with TGF-beta1 in the presence or absence of SD-208, a specific inhibitor of the TGF-beta type I receptor ALK5, or ALK5 small interfering (si)RNA. TGF-beta-signaling pathways were characterized by analysis of phosphorylated Smad2 or -1/5/8 proteins and TGF-beta target genes (PAI-1, fibronectin, CTGF, Smad7, and Id1) and protein (fibronectin). Results. ALK5 was expressed in both cell types, whereas ALK1 was exclusively expressed in endothelial cells. In endothelial cells, TGF-beta induced Smad2 phosphorylation at high concentrations, which was efficiently blocked by ALK5 inhibition. In contrast, in pericytes, Smad2 phosphorylation was rapidly induced at low concentrations of TGF-beta. The ALK1-Smad1/5/8 pathway was activated by TGF-beta in endothelial cells only. TGF-beta caused ALK5-mediated upregulation of PAI-1, Smad7, and fibronectin and in pericytes at lower TGF-beta concentrations than in endothelial cells. CTGF mRNA expression was induced only in pericytes. Fibronectin protein was confirmed to be regulated by TGF-beta in both cell types. Conclusions. TGF-beta signaling in retinal endothelial cells and pericytes show that these cells, and in particular the pericytes, have the essential characteristics to allow for a role of TGF-beta in BL thickening in preclinical diabetic retinopathy.

Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway

BackgroundTransforming growth factor-beta (TGF-beta) is known to exert multiple regulatory functions in the human placenta, including inhibition of estrodial production. We have previously reported that TGF-beta1 decreased aromatase mRNA levels in human trophoblast cells. The objective of this study was to investigate the molecular mechanisms underlying the regulatory effect of TGF-beta1 on aromatase expression.MethodsTo determine if TGF-beta regulates aromatase gene transcription, several reporter constructs containing different lengths of the placental specific promoter of the human aromatase gene were generated. JEG-3 cells were transiently transfected with a promoter construct and treated with or without TGF-beta1. The promoter activity was measured by luciferase assays. To examine the downstream signaling molecule mediating the effect of TGF-beta on aromatase transcription, cells were transiently transfected with dominant negative mutants of TGF-beta type II (TbetaRII) and type I receptor (ALK5) receptors before TGF-beta treatment. Smad2 activation was assessed by measuring phophorylated Smad2 protein levels in cytosolic and nuclear fractions. Smad2 expression was silenced using a siRNA expression construct. Finally, aromatase mRNA half-life was determined by treating cells with actinomycin D together with TGF-beta1 and measuring aromatase mRNA levels at various time points after treatment.Results and DiscussionTGF-beta1 inhibited the aromatase promoter activity in a time- and dose-dependent manner. Deletion analysis suggests that the TGF-β1 response element resides between -422 and -117 nucleotides upstream from the transcription start site where a Smad binding element was found. The inhibitory effect of TGF-beta1 was blocked by dominant negative mutants of TbetaRII and ALK5. TGF-beta1 treatment induced Smad2 phosphorylation and translocation into the nucleus. On the other hand, knockdown of Smad2 expression reversed the inhibitory effect of TGF-beta1 on aroamtase transcription. Furthermore, TGF-beta1 accelerated the degradation of aromatase mRNA.ConclusionOur results demonstrate that TGF-beta1 exerts regulatory effects on aromatase gene at both transcriptional and post-transcriptional levels. The transcriptional regulation of aromatase gene by TGF-beta1 is mediated by the canonical TGF-beta pathway involving TbetaRII, ALK5 and Smad2. These findings further support the role of TGF-beta1 in regulating human placental functions and pregnancy.

Molecular Sequaele of Activin A-Dependent Osteoblast Inhibition in Myeloma.

Abstract 1789Poster Board I-815Understanding the pathogenesis of cancer-related bone disease is critical to the discovery of new therapies. The development of osteolytic lesions in multiple myeloma (MM) results from unopposed osteoclast activity due to decreased osteoblast (OB) function. We recently demonstrated that activin A contributes to MM-mediated OB inhibition. The availability of a clinical-grade activin A inhibitor that reduces tumor burden by restoring the bone structure in vivo, underscores the relevance of this pathway in the development of MM-bone disease. Here, we characterize the signaling pathway mediating OB inhibition by activin A. Activin A secretion by bone marrow stromal cells (BMSC) is enhanced in the presence of MM cells which was completely abrogated and using a transwell system. To identify the receptors and pathways involved in activin A secretion, we used neutralizing antibody against several integrins, CD40 and osteopontin. Only VLA4 neutralizing antibodies partially inhibited activin secretion by about 20% (range 10-30%, p<0.05). Since activin A promoter contains a cJUN-binding sequence, we explored the relations between JNK and activin. We show that upon MM cell binding to BMSC, stromal-derived JNK is phosphorylated and SP600125 (20 μM), a specific JNK inhibitor, impairs activin A secretion by BMSC both at basal conditions and in the presence of MM (40% and 54%, respectively; p<0.01). These results suggest that activin secretion requires JNK activation induced by cell-to-cell contact. We next investigated the mechanism underlying activin-mediated OB inhibition by assessing the activity of several signaling pathways critical to OB differentiation. First, we showed that activin A induces SMAD2 phosphorylation, while no effects were noted on SMAD1, β-catenin, pP65 and pERK. Then, using a doxycycline-inducible β-catenin system, we demonstrate that β-catenin activation overcomes activin A inhibition of OB differentiation. These results suggest that activin/SMAD2 and β-catenin modulate OB differentiation by affecting a common downstream target. We next evaluated the expression of two candidates, the transcription factors RUNX2 and DLX5. Only DLX5 expression was downregulated by exogenous activin A (5 fold decrease at 96 hours, p< 0.01). Specific inhibition of SMAD2 via shRNA-mediated knock-down upregulated basal DLX5 and ALP mRNA expression (1.5 and 4.7 fold increase respectively, p<0.05), and partially overcame activin A inhibitory effects. In turn, DLX5 knock-down abrogated OB differentiation without additive effects by exogenous activin A. The clinical relevance of DLX5 was confirmed by the strong correlation between its expression levels in BM biopsies from MM patients and activin A levels in BM serum. Finally, we demonstrate by in vitro and in-vivo studies that MM-mediated DLX5 inhibition is restored by treatment with RAP-011, the specific activin inhibitor. In conclusion, we show that MM cell engagement of BMSC enhances activin A secretion via adhesion-mediated JNK activation and activin A, in turn, inhibits osteoblast differentiation via SMAD2-dependent DLX5 downregulation. This study identifies a novel pathway relevant to OB differentiation and amenable to drug targeting. DisclosuresChauhan:Nereus Pharmaceuticals, Inc: Consultancy. Seehra:Acceleron Pharma: Employment. Anderson:Celgene : Research Funding; Novartis: Research Funding; Millennium: Research Funding. Scadden:Fate Therapeutics: Consultancy. Raje:Astrazeneca, Novartis, Celgene: Research Funding.

Breast Cancer-derived Factors Stimulate Osteoclastogenesis through the Ca2+/Protein Kinase C and Transforming Growth Factor-β/MAPK Signaling Pathways

Breast cancer commonly metastasizes to bone where its growth depends on the action of bone-resorbing osteoclasts. We have previously shown that breast cancer cells secrete factors able to directly stimulate osteoclastogenesis from receptor activator of nuclear factor kappaB ligand (RANKL)-primed precursors and that transforming growth factor-beta (TGFbeta) plays a permissive role in this process. Now, we evaluate the signaling events triggered in osteoclast precursors by soluble factors produced by MDA-MB-231 human breast carcinoma cells. In mouse bone marrow cultures and RAW 264.7 murine monocytic cells, MDA-MB-231-derived factors increased osteoclast number, size, and nucleation. These factors failed to induce Smad2 phosphorylation, and short interfering RNAs against Smad4 did not affect their ability to induce osteoclastogenesis. In contrast, MDA-MB-231 factors induced phosphorylation of p38 and ERK1/2, and pharmacological inhibitors against p38 (SB203580) and MEK1/2 (PD98059) impeded the osteoclastogenic effects of cancer-derived factors. Neutralizing antibodies against TGFbeta attenuated p38 activation, whereas activation of ERK1/2 was shortened in duration, but not decreased in amplitude. ERK1/2 phosphorylation induced by cancer-derived factors was blocked by MEK1/2 inhibitor, but not by Ras (manumycin A) or Raf (GW5074) inhibitors. Inhibition of protein kinase Calpha using Gö6976 prevented both ERK1/2 phosphorylation and osteoclast formation in response to MDA-MB-231-derived factors. Using microspectrofluorimetry of fura-2-AM-loaded osteoclast precursors, we have found that cancer-derived factors, similar to RANKL, induced sustained oscillations in cytosolic free calcium. The calcium chelator BAPTA prevented calcium elevations and osteoclast formation in response to MDA-MB-231-derived factors. Thus, we have shown that breast cancer-derived factors induce osteoclastogenesis through the activation of calcium/protein kinase Calpha and TGFbeta-dependent ERK1/2 and p38 signaling pathways.

An injury-induced cell model of early uterine fibroid development

OBJECTIVE: The etiology of uterine fibroids is unclear and there is no universally accepted model for this common condition often causing pain, bleeding and infertility. Recent studies indicate uterine fibroids are formed by accumulation of ECM rather than by cellular proliferation and that altered wound healing leading to fibrosis may be the underlying mechanism. Fibrogenesis is a complex process in which the transforming growth factor-beta (TGFb) pathway plays a major role. TGFb3 and an activator of TGFb, thrombospondin-1, were previously reported genes disregulated in uterine fibroids. DESIGN: In an in-vitro cell culture model of myometrial cells we characterized fibrotic pathways activated due to injury. MATERIALS AND METHODS: Smooth muscle cells from fresh myometrium obtained at hysterectomy from non-pregnant women were cultured in 10% serum until 70% confluent. Injury was simulated by 72 h serum-starvation (SS) followed by serum replacement (SB). Cells were evaluated for cell viability and examined by electron microscopy (EM) and confocal microscopy. We measured mRNA and/or protein expression of thrombospondin-1 after SB, and Smad3 phosphorylation after TGFb treatment. RESULTS: Cell viability after 72 h SS did not differ from controls. Cells stained positive for human α-SMA. EM showed 60-80 % of SS cells had notched nuclei and a decreased cellular/nuclear ratio characteristic of myofibroblasts, cells active in wound repair and fibrosis. Thrombospondin-1 mRNA and protein expression were increased within 2 h after SB and remained elevated. TGFb treatment induced Smad3 phosphorylation. CONCLUSIONS: We conclude that myometrial cells can be differentiated to myofibroblast-like cells and that wound healing and early fibrosis pathways with a potential role in early uterine fibroid development are inducible in this cell model. This model offers a promising approach for exploring the pathogenesis of uterine fibroids and for developing cell-based therapeutic strategies.

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.

Activin Receptor Signaling Regulates Prostatic Epithelial Cell Adhesion and Viability

Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum factor(s) in cell proliferation. Using prostatic cancer cell (PCC) lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII), as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS). Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic fibroblast growth factor was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-15 expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer growth and metastasis.

T cells regulate interstitial lung disease via expression of integrins (95.9)

Abstract Interstitial lung disease, a combination of pulmonary inflammation and fibrosis, is deadly and occurs idiopathically or as a complication of autoimmune rheumatic diseases (scleroderma, rheumatoid arthritis, poly- and dermatomyositis, lupus). T cells often accumulate in the lungs of such patients. Previous reports suggested that αV-containing integrins may activate TGF-β and facilitate fibrosis. Whether pulmonary T cells utilize such integrins in interstitial lung disease is not clear. Expression of integrins αVβ3 (ITGAVB3) or αVβ5 (ITGAVB5) on pulmonary T cells was observed by double-stain immunofluorescence confocal microscopy in six out of six patients with scleroderma lung disease, as well as in the animal models of pulmonary CCL18 overexpression that manifests in selective T lymphocytic infiltration of the lungs and T cell-dependent pulmonary fibrosis. Flow cytometry of bronchoalveolar lavage T cells confirmed expression of integrins on more than 10% (up to 50%) of pulmonary T cells in 14 of 25 scleroderma patients and in all CCL18-overexpressing mice; Q-PCR experiments confirmed elevated integrin mRNAs in purified BAL T cells. There was no expression of ITGAVB3 or ITGAVB5 on T cells from the lungs of healthy volunteers or control mice. Injections of anti-ITGB3 blocking antibody or genetic deficiency of ITGB3 protected mice from CC18-induced inflammation and fibrosis. Jurkat cells that were forced to overexpress ITGAVB3 or ITGAVB5 induced Smad2 phosphorylation and nuclear translocation, and increases in collagen and α-smooth muscle actin expression in co-cultures with primary lung fibroblasts. Blocking anti-TGF-β antibodies attenuated such regulation in cell co-cultures. Thus, expression of integrins on pulmonary T cells contributes to interstitial lung disease by promoting T cell infiltration, TGF-β activation, and fibrosis.

Ionizing Radiation Shifts the PAI-1/ID-1 Balance and Activates Notch Signaling in Endothelial Cells

Transforming growth factor-beta (TGF-beta) and Notch signaling pathways are important regulators of vascular homeostasis and vessel remodeling; mutations in these pathways can lead to vascular disorders. Similar vascular phenotypes develop in the normal tissues of cancer patients as a long-term effect of radiotherapy. Irradiation most severely affects the capillaries, which become leaky and dilated and might eventually rupture. To investigate the mechanism of such capillary damage, we studied the effect of TGF-beta and Notch signaling in microvascular endothelial cells. Human microvascular endothelial cells were irradiated with 5 or 10 Gy and activation of TGF-beta and Notch signaling pathways was assessed by biochemical methods and a cell migration assay. Ionizing radiation induced Smad2 phosphorylation and nuclear translocation and increased mRNA and protein expression of the activin-like kinase 5 (ALK5) target gene plasminogen activator inhibitor-1 (PAI-1). At the same time, we observed diminished Smad1/5/8 activation and downregulation of the ALK1 downstream target, inhibitor of DNA binding-1 (ID-1). We also measured an upregulation of the Notch ligand Jagged-1 and the target gene Hey1. Decreased inhibitor of DNA binding-1 levels coincided with a reduced ability of the cells to migrate. Ionizing radiation shifts the balance from ALK1 to ALK5 signaling and activates the Notch pathway in endothelial cells. This combination of anti-angiogenic signals contributes to reduced cell migration after irradiation.

Transforming Growth Factor-β Activity in Commercially Available Pasteurized Cow Milk Provides Protection against Inflammation in Mice

Cow milk contains a large amount of an immunoregulatory cytokine, transforming growth factor-β (TGFβ). The present study investigated whether commercially available pasteurized cow milk retains TGFβ activity both in vitro and in vivo. Some commercial cow milk increased TGFβ/Smad-responsive reporter activity and induced Smad2 phosphorylation and the transcription of the TGFβ/Smad target genes TGFβ itself and Smad7 in vitro. Mice treated orally with 500 μL of cow milk containing TGFβ (3 μg/L) daily for 2 wk had increased phosphorylation of Smad2 and TGFβ and Smad7 mRNA expression in the intestine. These mice also had significantly greater serum TGFβ concentrations than the mice treated orally with PBS. Furthermore, oral administration of 500 μL of cow milk containing TGFβ (3 μg/L) daily for 2 wk before the induction of dextran sodium sulfate colitis and lipopolysaccharide-induced endotoxemia ameliorated tissue damage and mortality, respectively, in mice. These in vivo effects of cow milk were abrogated by the simultaneous administration of TGFβ type I receptor kinase inhibitor with the cow milk, and they were not observed after the oral administration of cow’s milk containing little TGFβ. In humans, 1 oral challenge of 10 mL/kg cow milk containing TGFβ (3 μg/L) increased the plasma TGFβ concentrations at 4 h after the challenge. Thus, some commercially available pasteurized cow milk retains TGFβ activity, which may be able to provide protection against experimental colitis and endotoxemia associated with increased intestinal and circulating TGFβ levels.

476 POSTER Involvement of the helix-loop-helix protein ID-1 in granulocytic differentiation of human myeloblastic leukemia cells

Transforming growth factor-β (TGF-β) and Notch signaling pathways are important regulators of vascular homeostasis and vessel remodeling; mutations in these pathways can lead to vascular disorders. Similar vascular phenotypes develop in the normal tissues of cancer patients as a long-term effect of radiotherapy. Irradiation most severely affects the capillaries, which become leaky and dilated and might eventually rupture. To investigate the mechanism of such capillary damage, we studied the effect of TGF-β and Notch signaling in microvascular endothelial cells.Human microvascular endothelial cells were irradiated with 5 or 10 Gy and activation of TGF-β and Notch signaling pathways was assessed by biochemical methods and a cell migration assay.Ionizing radiation induced Smad2 phosphorylation and nuclear translocation and increased mRNA and protein expression of the activin-like kinase 5 (ALK5) target gene plasminogen activator inhibitor-1 (PAI-1). At the same time, we observed diminished Smad1/5/8 activation and downregulation of the ALK1 downstream target, inhibitor of DNA binding-1 (ID-1). We also measured an upregulation of the Notch ligand Jagged-1 and the target gene Hey1. Decreased inhibitor of DNA binding-1 levels coincided with a reduced ability of the cells to migrate.Ionizing radiation shifts the balance from ALK1 to ALK5 signaling and activates the Notch pathway in endothelial cells. This combination of anti-angiogenic signals contributes to reduced cell migration after irradiation.