Steffen K. Meurer

Endoglin Differentially Modulates Antagonistic Transforming Growth Factor-β1 and BMP-7 Signaling

Transforming growth factor-β1 (TGF-β1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-β type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-β/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-β1 signaling and the role of endoglin for both pathways in endoglin-deficient L6E9 cells. In this myoblastic cell line, TGF-β1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-β1) are two examples. TGF-β1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-β1-induced collagen I expression and (CAGA)12-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-β1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-β signaling.

UNLABELLED Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. CONCLUSION In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.

BMP-7 as antagonist of organ fibrosis.

Fibrosis is a scarring process that is a common feature of chronic organ injury. It is characterized by elevated activity of transforming growth factor-beta resulting in increased and altered deposition of extracellular matrix and other fibrosis-associated proteins. Recent work has demonstrated that bone morphogenetic protein-7 blocks transforming growth factor-beta signaling. Moreover, member of the CCN family, Endoglin, Sclerostin, Sclerostin domain-containing proteins, Gremlin, Noggin, Chordin, and Kielin/Chordin-like protein influence the biological activity of both cytokines. As a consequence, they modulate cellular proliferation, migration, adhesion and extracellular matrix production. This tight protein network consisting of transforming growth factor-betas, bone morphogenetic proteins and various binding partners includes potential novel molecular targets and biomarkers useful for prognostication, disease monitoring and therapy. We here summarize recent advances in understanding bone morphogenetic protein-7 function and signaling and the current attempts to use this critical modulator as a pharmacological device to reverse transforming growth factor-beta-induced fibrogenesis.

Expression and functional analysis of endoglin in isolated liver cells and its involvement in fibrogenic Smad signalling.

Endoglin is an accessory component of the TGF-β-binding receptor complex that differentially modulates TGF-β and BMP responses. The existence of two splice variants L- and S-endoglin which differ in their cytoplasmic domain has already been shown in human and mice. Endoglin is located on the cell surfaces of cultured hepatic stellate cells and transdifferentiated myofibroblasts suggesting that this receptor might be associated with the profibrogenic attributes of these liver cell subpopulations. We now show that endoglin expression is increased in transdifferentiating hepatic stellate cells and in two models of liver fibrosis (i.e. bile duct ligation and carbon tetrachloride model) and further detectable in cultured portal fibroblasts representing another important fibrogenic cell type but not in hepatocytes. In respect to TGF-β1-signalling, we demonstrate that endoglin interacts with and is phosphorylated by TβRII. In hepatic stellate cells, TGF-β1 upregulates endoglin expression most likely via the ALK5 pathway and requires the SP1 transcription factor. We further identified a novel rat splice variant that is structurally and functionally different from that identified in human and mouse. Transient overexpression of endoglin resulted in a strong increase of TGF-β1-driven Smad1/5 phosphorylation and α-smooth muscle actin expression in a hepatic stellate cell line. In supernatants of respective cultures, we could detect the ectodomain of endoglin suggesting that shedding is a further key process involved in the regulation of this surface receptor.

Overexpression of Endoglin Modulates TGF-β1-Signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

Hepatic stellate cells (HSCs) play a major role in the pathogenesis of liver fibrosis. Working on primary HSCs requires difficult isolation procedures; therefore we have generated and here characterize a mouse hepatic stellate cell line expressing GFP under control of the collagen 1(I) promoter/enhancer. These cells are responsive to pro-fibrogenic stimuIi, such as PDGF or TGF-β1, and are able to activate intracellular signalling pathways including Smads and MAP kinases. Nevertheless, due to the basal level of activation, TGF-β1 did not significantly induce GFP expression contrasting the TGF-β1 regulated endogenous collagen I expression. We could demonstrate that the accessory TGF-β-receptor endoglin, which is endogenously expressed at very low levels, has a differential effect on signalling of these cells when transiently overexpressed. In the presence of endoglin activation of Smad1/5/8 was drastically enhanced. Moreover, the phosphorylation of ERK1/2 was increased, and the expression of vimentin, α-smooth muscle actin and connective tissue growth factor was upregulated. Endoglin induced a slight increase in expression of the inhibitor of differentiation-2 while the amount of endogenous collagen type I was reduced. Therefore, this profibrogenic cell line with hepatic stellate cell origin is not only a promising novel experimental tool, which can be used in vivo for cell tracing experiments. Furthermore it allows investigating the impact of various regulatory proteins (e.g. endoglin) on profibrogenic signal transduction, differentiation and hepatic stellate cell biology.

TGF-beta1 Does Not Induce Senescence of Multipotent Mesenchymal Stromal Cells and Has Similar Effects in Early and Late Passages

Transforming growth factor-beta 1 (TGF-β1) stimulates a broad range of effects which are cell type dependent, and it has been suggested to induce cellular senescence. On the other hand, long-term culture of multipotent mesenchymal stromal cells (MSCs) has a major impact on their cellular physiology and therefore it is well conceivable that the molecular events triggered by TGF-β1 differ considerably in cells of early and late passages. In this study, we analyzed the effect of TGF-β1 on and during replicative senescence of MSCs. Stimulation with TGF-β1 enhanced proliferation, induced a network like growth pattern and impaired adipogenic and osteogenic differentiation. TGF-β1 did not induce premature senescence. However, due to increased proliferation rates the cells reached replicative senescence earlier than untreated controls. This was also evident, when we analyzed senescence-associated DNA-methylation changes. Gene expression profiles of MSCs differed considerably at relatively early (P 3 - 5) and later passages (P 10). Nonetheless, relative gene expression differences provoked by TGF-β1 at individual time points or in a time course dependent manner (stimulation for 0, 1, 4 and 12 h) were very similar in MSCs of early and late passage. These results support the notion that TGF-β1 has major impact on MSC function, but it does not induce senescence and has similar molecular effects during culture expansion.

Genetic characteristics of the human hepatic stellate cell line LX-2.

The human hepatic cell line LX-2 has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds. It was originally generated by immortalisation with the Simian Vacuolating Virus 40 (SV40) transforming (T) antigen and subsequent propagation in low serum conditions. Although this immortalized line is used in an increasing number of studies, detailed genetic characterisation has been lacking. We here have performed genetic characterisation of the LX-2 cell line and established a single-locus short tandem repeat (STR) profile for the cell line and characterized the LX-2 karyotype by several cytogenetic and molecular cytogenetic techniques. Spectral karyotyping (SKY) revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers. In addition, various subclonal and single cell aberrations were detected. Our study provides criteria for genetic authentication of LX-2 and offers insights into the genotype changes which might underlie part of its phenotypic features.

TGF-β stimulation in human and murine cells reveals commonly affected biological processes and pathways at transcription level

BackgroundThe TGF-β signaling pathway is a fundamental pathway in the living cell, which plays a key role in many central cellular processes. The complex and sometimes contradicting mechanisms by which TGF-β yields phenotypic effects are not yet completely understood. In this study we investigated and compared the transcriptional response profile of TGF-β1 stimulation in different cell types. For this purpose, extensive experiments are performed and time-course microarray data are generated in human and mouse parenchymal liver cells, human mesenchymal stromal cells and mouse hematopoietic progenitor cells at different time points. We applied a panel of bioinformatics methods on our data to uncover common patterns in the dynamic gene expression response in respective cells.ResultsOur analysis revealed a quite variable and multifaceted transcriptional response profile of TGF-β1 stimulation, which goes far beyond the well-characterized classical TGF-β1 signaling pathway. Nonetheless, we could identify several commonly affected processes and signaling pathways across cell types and species. In addition our analysis suggested an important role of the transcription factor EGR1, which appeared to have a conserved influence across cell-types and species. Validation via an independent dataset on A549 lung adenocarcinoma cells largely confirmed our findings. Network analysis suggested explanations, how TGF-β1 stimulation could lead to the observed effects.ConclusionsThe analysis of dynamical transcriptional response to TGF-β treatment experiments in different human and murine cell systems revealed commonly affected biological processes and pathways, which could be linked to TGF-β1 via network analysis. This helps to gain insights about TGF-β pathway activities in these cell systems and its conserved interactions between the species and tissue types.

Endoglin in liver fibrogenesis: Bridging basic science and clinical practice

Endoglin, also known as cluster of differentiation CD105, was originally identified 25 years ago as a novel marker of endothelial cells. Later it was shown that endoglin is also expressed in pro-fibrogenic cells including mesangial cells, cardiac and scleroderma fibroblasts, and hepatic stellate cells. It is an integral membrane-bound disulfide-linked 180 kDa homodimeric receptor that acts as a transforming growth factor-β (TGF-β) auxiliary co-receptor. In humans, several hundreds of mutations of the endoglin gene are known that give rise to an autosomal dominant bleeding disorder that is characterized by localized angiodysplasia and arteriovenous malformation. This disease is termed hereditary hemorrhagic telangiectasia type I and induces various vascular lesions, mainly on the face, lips, hands and gastrointestinal mucosa. Two variants of endoglin (i.e., S- and L-endoglin) are formed by alternative splicing that distinguishes from each other in the length of their cytoplasmic tails. Moreover, a soluble form of endoglin, i.e., sol-Eng, is shedded by the matrix metalloprotease-14 that cleaves within the extracellular juxtamembrane region. Endoglin interacts with the TGF-β signaling receptors and influences Smad-dependent and -independent effects. Recent work has demonstrated that endoglin is a crucial mediator during liver fibrogenesis that critically controls the activity of the different Smad branches. In the present review, we summarize the present knowledge of endoglin expression and function, its involvement in fibrogenic Smad signaling, current models to investigate endoglin function, and the diagnostic value of endoglin in liver disease.

PDGF-D signaling in portal myofibroblasts and hepatic stellate cells proves identical to PDGF-B via both PDGF receptor type α and β.

UNLABELLED Platelet-derived growth factor-D (PDGF-D) is one member of PDGF growth factors and known to signal by binding to and activating its cognate receptor type β (PDGFR-β). Beside PDGF-B, PDGF-D is a potent growth factor for stellate cell growth and proliferation and therefore potentiates the extracellular matrix deposition in liver fibrogenesis. We aimed to explore the signaling and molecular mechanisms of PDGF-D in liver fibrogenesis using the primary liver portal myofibroblasts and hepatic stellate cells. Unexpectedly we found PDGF-D to bind to PDGFR-α, thus inducing receptor endocytosis and decreasing the amount of PDGFR-α significantly. PDGF-D activates PDGFR-α specific tyrosine 754 and -1018 phosphorylation and CrkII, the adaptor protein that is specifically recruited by activated PDGFR-α. As a novel finding we could also demonstrate that recombinant PDGFR-α-Fc chimera homodimer is able to bind PDGF-D and thus prevent PDGF-D signaling. PDGF-D does induce individual PDGFR-β specific tyrosine phosphorylation similar to the PDGF-B. Additionally, PDGF-D enhances extracellular matrix accumulation comparable to the PDGF-B isoform. CONCLUSION PDGF-D signaling in pMF and HSC is identical to that of PDGF-B by binding to both PDGFR-α and -β.