TGF-β Target Genes Research Articles

Redox control of p53 in the transcriptional regulation of TGF-β1 target genes through SMAD cooperativity

Transforming growth factor-β1 (TGF-β1) regulates the tissue response to injury and is the principal driver of excessive scarring leading to fibrosis and eventual organ failure. The TGF-β1 effectors SMAD3 and p53 are major contributors to disease progression. While SMAD3 is an established pro-fibrotic factor, the role of p53 in the TGF-β1-induced fibrotic program is not clear. p53 gene silencing, genetic ablation/subsequent rescue, and pharmacological inhibition confirmed that p53 was required for expression of plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1 target gene and a key causative element in fibrotic disorders. TGF-β1 regulated p53 activity by stimulating p53Ser15 and 9 phosphorylation and acetylation, promoting interactions with activated SMADs and subsequent binding of p53/SMAD3 to the PAI-1 promoter in HK-2 human renal tubular epithelial cells and HaCaT human keratinocytes. Immunohistochemistry revealed prominent co-induction of SMAD3, p53 and PAI-1 in the tubular epithelium of the obstructed kidney consistent with a potential in vivo role for p53 and SMADs in TGF-β1-driven renal fibrosis. TGF-β1-initiated phosphorylation of p53Ser15 and up-regulation of expression of several pro-fibrotic genes, moreover, was dependent on the rapid generation of reactive oxygen species (ROS). shRNA silencing of the p22Phox subunit of NADP(H) oxidases in HK-2 cells partially attenuated (over 50%) p53Ser15 phosphorylation and PAI-1 induction. These studies highlight the role of free radicals in p53 activation and subsequent pro-fibrotic reprogramming by TGF-β1 via the SMAD3-p53 transcriptional axis. Present findings provide a rationale for therapeutic targeting of SMAD3-p53 in aberrant TGF-β1 signaling associated with renal fibrosis.

Stimulation of the soluble guanylate cyclase (sGC) inhibits fibrosis by blocking non-canonical TGFβ signalling

ObjectivesWe have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between...

Oxidative exposure impairs TGF-β pathway via reduction of type II receptor and SMAD3 in human skin fibroblasts

Exposure to oxidants results in cellular alterations that are implicated in aging and age-associated diseases. Here, we report that brief, low-level oxidative exposure leads to long-term elevation of cellular reactive oxygen species (ROS) levels and oxidative damage in human skin fibroblasts. Elevated ROS impairs the transforming growth factor-β (TGF-β) pathway, through reduction of type II TGF-β receptor (TβRII) and SMAD3 protein levels. This impairment results in reduced expression of connective tissue growth factor (CTGF/CCN2) and type I collagen, which are regulated by TGF-β. Restoration of TβRII and SMAD3 together, but not separately, reinstates TGF-β signaling and increases CTGF/CCN2 and type I collagen levels. Treatment with the anti-oxidant N-acetylcysteine reduces ROS elevation and normalizes TGF-β signaling and target gene expression. These data reveal a novel linkage between limited oxidant exposure and altered cellular redox homeostasis that results in impairment of TGF-β signaling. This linkage provides new insights regarding the mechanism by which aberrant redox homeostasis is coupled to decline of collagen production, a hallmark of human skin aging.

Genome-Wide Transcriptional Analysis of Differentially Expressed Genes in Diabetic, Healing Corneal Epithelial Cells: Hyperglycemia-Suppressed TGFβ3 Expression Contributes to the Delay of Epithelial Wound Healing in Diabetic Corneas

Patients with diabetes mellitus (DM) may develop corneal complications and delayed wound healing. The aims of this study are to characterize the molecular signatures and biological pathways leading to delayed epithelial wound healing and to delineate the involvement of TGFβ3 therein. Genome-wide cDNA microarray analysis revealed 1,888 differentially expressed genes in the healing epithelia of normal (NL) versus type 1 DM rat corneas. Gene ontology and enrichment analyses indicated TGFβ signaling as a major altered pathway. Among three TGFβ isoforms, TGF-β1 and β3 were upregulated in response to wounding in NL corneal epithelial cells (CECs), whereas the latter was greatly suppressed by hyperglycemia in rat type 1 and 2 and mouse type 1 DM models. Functional analysis indicated that TGF-β3 contributed to wound healing in NL corneas. Moreover, exogenously added TGF-β3 accelerated epithelial wound closure in type 2 rat and type 1 mouse DM corneas via Smad and PI3K-AKT signaling pathways, autoregulation, and/or upregulation of Serpine1, a well-known TGFβ target gene. Taken together, our study for the first time provides a comprehensive list of genes differentially expressed in the healing CECs of NL versus diabetic corneas and suggests the therapeutic potential of TGF-β3 for treating corneal and skin wounds in diabetic patients.

Differential regulation of Id1 expression by TGF-β1 in HepG2 cells

Introduction: In early stages of hepatocellular carcinoma (HCC) TGF-β is proposed to have a tumour suppressive function whereas in advanced HCC it acts as a tumor promoter [1]. However, the relevance of a potential TGF-β1 target gene, i.e. Id1, is discussed controversial during HCC development and progression [2 – 4]. In HCC cell lines expressing various types of TGF-β receptors and being growth inhibited by TGF-β1, Id1 is suppressed by this ligand [5]. In order to refine the response pattern to TGF-β1 and identify putative regulatory circuits in HCC cells, we analysed the gene activation pattern in the TGF-β responsive HepG2 cell line. Methods and results: The time dependent mRNA expression in response to TGF-β1 was analysed by microarray analysis. 166 candidate genes were found to be differentially regulated during the time course. These were grouped in five clusters according to their expression kinetics. Candidate genes were validated by qRT-PCR and the corresponding protein expression and activation of signalling intermediates was confirmed by Western blot analysis. Most genes were localized in the immediate early cluster including pathway inhibitors, i.e. Smad6, Smad7, Skil, and Id1. The latter showed a biphasic expression, being immediate early induced (2 hrs) and thereafter repressed (4 hrs), an expression pattern that was also found in hepatic stellate cells. The function of Id1 was elucidated by transient overexpression in HepG2 and found to influence Connective Tissue Growth Factor protein expression and Smad activation. Induction and repression were direct responses since cycloheximide failed to abrogate the observed effects on Id1 expression. In contrast the ALK5 inhibitor SB-431542 partially blocked induction and repression of Id1. Conclusion: Id1 is an important target gene of TGF-β1-signalling in hepatoma cells which are sensitive to TGF-β1 and express ALK5 since this receptor is crucial for the complex regulation of this target gene.

Impact of soluble Endoglin on TGF-β1 mediated immediate early responses in HepG2 cells

Introduction: Expression of Endoglin has been demonstrated in all cells of the liver except hepatocytes [1]. However, data published with respect to the hepatic function of Endoglin is patchy [2]. Nevertheless, there is a wealth of data detecting the soluble form of this receptor in the serum under different pathologic situations affecting the liver, e.g. cystic fibrosis associated liver disease [3]. We have previously shown that Endoglin is highly expressed in hepatic stellate cells [4]. Upon shedding from the HSC surface, this receptor might act not only in an autocrine but also in a paracrine fashion on signalling processes mediated by TGF-β1 in hepatocytes. Methods and results: Transient transfection of HepG2 with a expression vector encoding soluble rat endoglin (solEng) results in secretion of high amounts of dimeric solEng in the cell culture supernatant. solEng can be co-precipitated with TGF-β1. As a mutual consequence of ligand binding/sequestration, the luciferase activity in response to TGF-β1 (measured by the (CAGA)12-MLP Luc reporter) and to BMP-2 (measured by the (BRE)2-MLP Luc reporter) is reduced. In addition, we find in Western blot experimentation that the expression of the TGF-β1 target genes connective tissue growth factor (CTGF), JunB, p21, Id1, and Id2 is differentially modulated under basal and TGF-β1-induced conditions. Upstream, the TGF-β1 type II receptor as well as the activation of the two Smad branches, i.e. Smad2 and Smad1/Smad5, but not Smad3 is affected by solEng. Conclusion: These results underscore the relevance of solEng in the liver not only as a serum marker but also as a modulator of TGF-β superfamily ligand-signaling in hepatocytes. The fact that TGF-β1 responses are modulated differentially points towards a more complex function of soling in addition to ligand sequestration.

Zinc Finger Protein 451 Is a Novel Smad Corepressor in Transforming Growth Factor-β Signaling

ZNF451 is a transcriptional cofactor localized to promyelocytic leukemia bodies. Here, we present evidence demonstrating that ZNF451 physically interacts with Smad3/4 and functionally inhibits TGF-β signaling. Increased expression of ZNF451 attenuates TGF-β-induced growth inhibitory and gene transcriptional responses, whereas depletion of ZNF451 enhances TGF-β responses. Mechanistically, ZNF451 blocks the ability of Smad3/4 to recruit p300 in response to TGF-β, which causes reduction of histone H3K9 acetylation on the promoters of TGF-β target genes. Taken together, ZNF451 acts as a transcriptional corepressor for Smad3/4 and negatively regulates TGF-β signaling.

Angiotensin II–dependent TGF-β signaling contributes to Loeys-Dietz syndrome vascular pathogenesis

Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. LDS is caused by heterozygous missense mutations in either TGF-β receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-β signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-β signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-β in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-β signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-β target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-β1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-β1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-β signaling contributes to postnatal aneurysm progression in LDS.

Deletion of the Transforming Growth Factor β Receptor Type II Gene in Articular Chondrocytes Leads to a Progressive Osteoarthritis‐like Phenotype in Mice

While transforming growth factor β (TGFβ) signaling plays a critical role in chondrocyte metabolism, the TGFβ signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFβ signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling. TGFβ receptor type II (TGFβRII)-conditional knockout (KO) (TGFβRII(Col2ER)) mice were generated by breeding TGFβRII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFβ signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFβ signaling, TGFβRII/matrix metalloproteinase 13 (MMP-13)- and TGFβRII/ADAMTS-5-double-KO mice were generated and analyzed. Inhibition of TGFβ signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFβRII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFβ signaling. Treatment of TGFβRII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression. Mmp13 and Adamts5 are critical downstream target genes involved in the TGFβ signaling pathway during the development of OA.

SPOCK1 is a novel transforming growth factor-β target gene that regulates lung cancer cell epithelial-mesenchymal transition

Lung cancer is the leading cause of cancer related death worldwide and the prognosis is still poor with 5-year survival of approximately 15%. Metastasis is the leading cause of death by cancer. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Here, we identified that SPARC/osteonectin, cwcv and kazal-like domains proteoglycan 1 (SPOCK1) is a novel transforming growth factor-β1 (TGF-β) target gene that regulates lung cancer cell EMT. TGF-β has been reported as a major inductor of EMT. We observed that the expression of SPOCK1 in lung cancer tumor tissues is significantly higher than matched normal lung tissues. Moreover, the expression of SPOCK1 was also significantly higher in metastasis tumor tissues than non-metastasis tumor tissues. Levels of SPOCK1 mRNA were increased among patients with shorter disease-free survival times, indicating the potential role of SPOCK1 in lung cancer progression and metastasis. Silencing SPOCK1 expression with endoribonuclease-prepared small interfering RNA (esiRNA) in lung cells inhibits lung cancer cell growth, colony formation and invasion in vitro. Interestingly, ectopic expression of SPOCK1 in epithelial lung cancer cells induced EMT with increased expression of the mesenchymal marker Vimentin and decreased expression of epithelial marker E-cadherin. We also found that the expression of SPOCK1 was increased under treatment of TGF-β, indicating that SPOCK1 is a novel downstream target of TGF-β. Taken together, our study showed that SPOCK1 is a novel metastasis related biomarker in lung cancer and may be new diagnostic and therapeutic target for lung cancer.

Abstract IA02: Tumor plasticity and reactive stroma in breast cancer metastasis

Abstract Metastasis represents the most devastating stage of cancer progression. The multi-step cascade of cancer metastasis is driven by both intrinsic changes of cancer cell properties, as well as the stromal reaction to tumor cells. Epithelial-mesenchymal transition (EMT) is a complex process that is associated with dramatic changes in cell adhesion, polarity, and migratory properties, and is intimately associated both with cell fate transitions during embryonic development and with the acquisition of invasive properties during early steps of cancer metastasis. We found that the transcription factor Elf5, a key regulator of alveologenesis in the mammary gland, regulates EMT in both mammary gland development and metastasis. This specific role for Elf5 in suppressing EMT and metastasis was uncovered via analyses of Elf5 conditional knockout animals, various in vitro and in vivo models of EMT and metastasis, an MMTV-neu transgenic model of mammary gland tumor progression, and clinical breast cancer samples. Furthermore, we demonstrate that this role of Elf5 is mediated by the direct transcriptional repression of Snail2/Slug, a master regulator of mammary stem cells and a known inducer of EMT. These findings establish a broad molecular function of Elf5 that extends from being a key cell lineage regulator during normal mammary gland development to serving as a suppressor of EMT and metastasis in breast cancer. The growth of disseminated tumor cells at distant organs represents a critical final stage of metastasis development. Bone metastasis is a frequent occurrence in breast cancer, affecting more than 70% of late stage cancer patients. Increasing evidence suggest that the development of bone metastasis relies on the positive feedbacks from stromal components of the bone microenvironment. Using in vivo imaging technology, we showed that TGFβ is released from bone matrix upon bone destruction, and signals to breast cancer to further enhance their malignancy in developing bone metastasis. We furthered identified Jagged1 as a TGFβ target genes in tumor cells that engaged bone stromal cells through the activation of Notch signaling to provide a positive feedback to promote tumor growth and to activate osteoclast differentiation. More recently, we identified osteoclast-derived miRNAs as biomarkers and therapeutic targets of osteolytic bone metastasis. Importantly, pharmaceutical inhibitors against TGFβ and Notch signaling pathways and miRNAs blocking osteoclast differentiation significantly reduce the development of bone metastasis, suggesting novel avenues for clinical management of skeletal complications of breast cancer. Citation Format: Yibin Kang. Tumor plasticity and reactive stroma in breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA02.

Syndecan-2 Exerts Antifibrotic Effects by Promoting Caveolin-1–mediated Transforming Growth Factor-β Receptor I Internalization and Inhibiting Transforming Growth Factor-β1 Signaling

Alveolar transforming growth factor (TGF)-β1 signaling and expression of TGF-β1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-β receptor TβRI inhibits TGF-β signaling and could attenuate development of experimental lung fibrosis. To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-β1 signaling in alveolar epithelial cells. Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2-dependent changes in epithelial cell TGF-β1 signaling, TGF-β1, and TβRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-β1 signaling and downstream expression of TGF-β1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1-dependent internalization of TGF-β1 and TβRI in alveolar epithelial cells, which inhibited TGF-β1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1-dependent TGF-β1 and TβRI internalization and inhibiting TGF-β1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TβRI degradation via endocytosis represent potential therapies for pulmonary fibrosis.

From TOR and SMAD, why HIF-1α can be bad

From TOR and SMAD, why HIF-1α can be bad

Sex Differences in the Pituitary Transforming Growth Factor-β1 System: Studies in a Model of Resistant Prolactinomas

Dopamine and estradiol interact in the regulation of lactotroph cell proliferation and prolactin secretion. Ablation of the dopamine D2 receptor gene (Drd2(-/-)) in mice leads to a sexually dimorphic phenotype of hyperprolactinemia and pituitary hyperplasia, which is stronger in females. TGF-β1 is a known inhibitor of lactotroph proliferation. TGF-β1 is regulated by dopamine and estradiol, and it is usually down-regulated in prolactinoma experimental models. To understand the role of TGF-β1 in the gender-specific development of prolactinomas in Drd2(-/-) mice, we compared the expression of different components of the pituitary TGF-β1 system, including active cytokine content, latent TGF-β-binding protein isoforms, and possible local TGF-β1 activators, in males and females in this model. Furthermore, we evaluated the effects of dopamine and estradiol administration to elucidate their role in TGF-β1 system regulation. The expression of active TGF-β1, latent TGF-β-binding protein isoforms, and several putative TGF-β1 activators evaluated was higher in male than in female mouse pituitary glands. However, Drd2(-/-) female mice were more sensitive to the decrease in active TGF-β1 content, as reflected by the down-regulation of TGF-β1 target genes. Estrogen and dopamine caused differential regulation of several components of the TGF-β1 system. In particular, we found sex- and genotype- dependent regulation of active TGF-β1 content and a similar expression pattern for 2 of the putative TGF-β1 activators, thrombospondin-1 and kallikrein-1, suggesting that these proteins could mediate TGF-β1 activation elicited by dopamine and estradiol. Our results indicate that (1) the loss of dopaminergic tone affects the pituitary TGF-β1 system more strongly in females than in males, (2) males express higher levels of pituitary TGF-β1 system components including active cytokine, and (3) estradiol negatively controls most of the components of the system. Because TGF-β1 inhibits lactotroph proliferation, we propose that the higher levels of the TGF-β1 system in males could protect or delay the development of prolactinomas in Drd2(-/-) male mice.

NPR2, SMAD2/3, BMP and GDF-9 expressions in human granulosa and cumulus cells during oocyte maturation

We compare expression of Natriuretic Peptide Receptor 2 (NPR2), SMAD2/3, bone morphogenetic proteins (BMP) receptor 2 (BMPR2), BMP15 and growth differentiation factor 9 (GDF-9) mRNA levels in human GCs and CCs in young and older IVF patients. NPR2 is an oocyte meiotic maturation inhibitor that maintains meiotic arrest. Its role in oocyte maturation is not well understood. SMAD2/3 are transcription factors that transduce TGF-β superfamily ligand signals from membrane-bound TGF-β receptors into nucleus, inducing TGF-β target gene expression associated with cumulus expansion.

HCV core-mediated activation of latent TGF-β via thrombospondin drives the crosstalk between hepatocytes and stromal environment

The mechanisms by which fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) develop during chronic hepatitis C virus (HCV) infection are not fully understood. We previously observed that HCV core protein induced a TGF-β-dependent epithelial mesenchymal transition, a process contributing to the promotion of cell invasion and metastasis by impacting TGF-β1 signalling. Here we investigated HCV core capacity to drive increased expression of the active form of TGF-β1n transgenic mice and hepatoma cell lines. We used an in vivo model of HCV core expressing transgenic mice. We observed that about 50% of genes deregulated by core protein expression were TGF-β1 target genes. Active TGF-β levels were increased in HCV core transgenic mouse livers. Overexpression of core protein in hepatoma cells increased active TGF-β levels in culture supernatants and induced Smad2/3 phosphorylation, thus reflecting activation of the TGF-β signaling pathway. Moreover, our data showed the implication of thrombospondin-1 in core-dependent TGF-β activation. Finally, hepatoma cells expressing HCV core could activate stellate cells in co-culture and this activation was TGF-β dependent. Collectively, these data delineate a novel paradigm where HCV may be related to liver pathogenesis through its ability to induce a local, intrahepatic TGF-β activation. They argue for a dual impact of HCV core on liver fibrosis and liver carcinogenesis: HCV core could act both as autocrine and paracrine factor modulating TGF-β responses within hepatocytes and in stromal environment through TGF-β activation.

Rho gtpase activating protein p190-B regulates hematopoietic stem cell self-renewal decisions

Hematopoietic stem cells (HSC) can self-renew or commit to differentiate into all blood cells. The signaling network that regulates these choices is poorly understood. We previously showed that p190-B loss, a negative regulator of Rho activity, enhanced long-term engraftment without altering HSC proliferation or survival or lineage development (Xu, Blood 2009), suggesting an effect on HSC fate decisions. Here, we compared single HSC divisions ex vivo by analyzing multilineage potential of the paired-daughter cells. Non-transplanted (NT) HSCs generated two multipotent daughter cells (self-renewing divisions). However, after secondary transplantation, wild-type (WT) HSCs generated one multipotent and one committed daughter cells (differentiating divisions). p190-B-/- HSCs maintained self-renewal divisions, indicating p190-B loss mediates higher probability of ‘stemness' inheritance through divisions to prevent HSC depletion following transplantation. Unexpectedly, p190-B loss does this by decreasing autocrine TGF-β. Active TGF-β protein levels, TGF-β target genes increased in WT but not in p190-B-/- HSCs upon transplantation relative to NT HSC. TGF-β signaling inhibitors restored self-renewal divisions of transplanted WT HSC as seen in vitro and in engraftment in vivo. TGF-β inhibitor treatment of transplanted mice significantly increased HSC frequency - without changing their proliferation - that yielded higher chimera in tertiary transplant compared to DMSO treated mice. Conversely, recombinant TGF-β1 changed non-transplanted HSC choice to differentiating divisions in vitro. Finally, p38MAPK activity mediated TGF-β effect on HSC fate decisions. Hence, HSC decisions to self-renew or differentiate are specified by a p190-B/TGF-β signaling pathway during HSC regeneration. This study uncovers a novel role for autocrine TGF-β in HSC fate decisions uncoupled from its role on HSC quiescence that may have important implications for regenerative medicine. Hematopoietic stem cells (HSC) can self-renew or commit to differentiate into all blood cells. The signaling network that regulates these choices is poorly understood. We previously showed that p190-B loss, a negative regulator of Rho activity, enhanced long-term engraftment without altering HSC proliferation or survival or lineage development (Xu, Blood 2009), suggesting an effect on HSC fate decisions. Here, we compared single HSC divisions ex vivo by analyzing multilineage potential of the paired-daughter cells. Non-transplanted (NT) HSCs generated two multipotent daughter cells (self-renewing divisions). However, after secondary transplantation, wild-type (WT) HSCs generated one multipotent and one committed daughter cells (differentiating divisions). p190-B-/- HSCs maintained self-renewal divisions, indicating p190-B loss mediates higher probability of ‘stemness' inheritance through divisions to prevent HSC depletion following transplantation. Unexpectedly, p190-B loss does this by decreasing autocrine TGF-β. Active TGF-β protein levels, TGF-β target genes increased in WT but not in p190-B-/- HSCs upon transplantation relative to NT HSC. TGF-β signaling inhibitors restored self-renewal divisions of transplanted WT HSC as seen in vitro and in engraftment in vivo. TGF-β inhibitor treatment of transplanted mice significantly increased HSC frequency - without changing their proliferation - that yielded higher chimera in tertiary transplant compared to DMSO treated mice. Conversely, recombinant TGF-β1 changed non-transplanted HSC choice to differentiating divisions in vitro. Finally, p38MAPK activity mediated TGF-β effect on HSC fate decisions. Hence, HSC decisions to self-renew or differentiate are specified by a p190-B/TGF-β signaling pathway during HSC regeneration. This study uncovers a novel role for autocrine TGF-β in HSC fate decisions uncoupled from its role on HSC quiescence that may have important implications for regenerative medicine.

Pharmacological Induction of Transforming Growth Factor-Beta1 in Rat Models Enhances Radiation Injury in the Intestine and the Heart

Radiation therapy in the treatment of cancer is dose limited by radiation injury in normal tissues such as the intestine and the heart. To identify the mechanistic involvement of transforming growth factor-beta 1 (TGF-β1) in intestinal and cardiac radiation injury, we studied the influence of pharmacological induction of TGF-β1 with xaliproden (SR 57746A) in rat models of radiation enteropathy and radiation-induced heart disease (RIHD). Because it was uncertain to what extent TGF-β induction may enhance radiation injury in heart and intestine, animals were exposed to irradiation schedules that cause mild to moderate (acute) radiation injury. In the radiation enteropathy model, male Sprague-Dawley rats received local irradiation of a 4-cm loop of rat ileum with 7 once-daily fractions of 5.6 Gy, and intestinal injury was assessed at 2 weeks and 12 weeks after irradiation. In the RIHD model, male Sprague-Dawley rats received local heart irradiation with a single dose of 18 Gy and were followed for 6 months after irradiation. Rats were treated orally with xaliproden starting 3 days before irradiation until the end of the experiments. Treatment with xaliproden increased circulating TGF-β1 levels by 300% and significantly induced expression of TGF-β1 and TGF-β1 target genes in the irradiated intestine and heart. Various radiation-induced structural changes in the intestine at 2 and 12 weeks were significantly enhanced with TGF-β1 induction. Similarly, in the RIHD model induction of TGF-β1 augmented radiation-induced changes in cardiac function and myocardial fibrosis. These results lend further support for the direct involvement of TGF-β1 in biological mechanisms of radiation-induced adverse remodeling in the intestine and the heart.

OP0016 Tribbles homolog 3 mediates the stimulatory effects of tgf-beta on fibroblast activation and dermal fibrosis in systemic sclerosis

BackgroundTribbles Homolog 3 (TRB3), is a member of the family of pseudokinases called Tribbles which regulate activation of various intracellular signaling pathways with roles extending from mitosis and cell activation...

P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation

Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling.