Biomedical subjects

Lucia Wickert

Publications and source records attributed to Lucia Wickert.

6 recordsLinked to original sources

PubMed2006

Id1 is a critical mediator in TGF-beta-induced transdifferentiation of rat hepatic stellate cells.

Transforming growth factor (TGF)-beta is critically involved in the activation of hepatic stellate cells (HSCs) that occurs during the process of liver damage, for example, by alcohol, hepatotoxic viruses, or aflatoxins. Overexpression of the TGF-beta antagonist Smad7 inhibits transdifferentiation and arrests HSCs in a quiescent stage. Additionally, bile duct ligation (BDL)-induced fibrosis is ameliorated by introducing adenoviruses expressing Smad7 with down-regulated collagen and alpha-smooth muscle actin (alpha-SMA) expression. The aim of this study was to further characterize the molecular details of TGF-beta pathways that control the transdifferentiation process. In an attempt to elucidate TGF-beta target genes responsible for fibrogenesis, an analysis of Smad7-dependent mRNA expression profiles in HSCs was performed, resulting in the identification of the inhibitor of differentiation 1 (Id1) gene. Ectopic Smad7 expression in HSCs strongly reduced Id1 mRNA and protein expression. Conversely, Id1 overexpression in HSCs enhanced cell activation and circumvented Smad7-dependent inhibition of transdifferentiation. Moreover, knock-down of Id1 in HSCs interfered with alpha-SMA fiber formation, indicating a pivotal role of Id1 for fibrogenesis. Treatment of HSCs with TGF-beta1 led to increased Id1 protein expression, which was not directly mediated by the ALK5/Smad2/3, but the ALK1/Smad1 pathway. In vivo, Id1 expression and Smad1 phosphorylation were co-induced during fibrogenesis. In conclusion, Id1 is identified as TGF-beta/ALK1/Smad1 target gene in HSCs and represents a critical mediator of transdifferentiation that might be involved in hepatic fibrogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Animals↗

PubMed2005

Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC).

The platelet-derived growth factor (PDGF) family, which regulates many physiological and pathophysiological processes has recently been enlarged by two new members, the isoforms PDGF-C and -D. Little is known about the expression levels of these new members in hepatic fibrosis. We therefore investigated by quantitative real time PCR (Taqman) the mRNA expression profiles of all four PDGF isoforms in transdifferentiating primary cultured hepatic stellate cells (HSC), an in vitro model system of hepatic fibrogenesis, either with or without stimulation of the cells with PDGF-BB or TGF-beta1. All four isoforms were expressed in HSC transdifferentiating to myofibroblast-like cells (MFB) albeit with different profiles: while PDGF-A mRNA exhibited minor fluctuations only, PDGF-B was rapidly down-regulated. In contrast, both PDGF-C and -D mRNA were strongly induced: PDGF-C up to 5 fold from day 2 to day 8 and PDGF-D up to 8 fold from day 2 to day 5 of culture. Presence of PDGF-DD in activated HSC was confirmed at the protein level by immunocytochemistry. Stimulation of HSC and MFB with PDGF-BB led to down-regulation of the new isoforms, whereas TGF-beta1 upregulated PDGF-A only. We further show that PDGF receptor-beta (PDGFR-beta) mRNA was rapidly upregulated within the first day of culture and was constantly expressed from day 2 on while the expression profile of PDGFR-alpha mRNA was very similar to that of PDGF-A during transdifferentiation. Given the dramatic changes in PDGF-C and -D expression, which may compensate for down-regulation of PDGF-B, we hypothesize that the new PDGF isoforms may fulfil specific functions in hepatic fibrogenesis.

Animals↗

PubMed2005

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts.

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.

Actins↗

PubMed2005

Transdifferentiation-dependent expression of alpha-SMA in hepatic stellate cells does not involve TGF-beta pathways leading to coinduction of collagen type I and thrombospondin-2.

Hepatic stellate cells (HSC) cultured on plastic spontaneously transdifferentiate to a myofibroblast-like cell type (MFB). This model system of hepatic fibrogenesis is characterized by phenotypic changes of the cells and increased matrix synthesis. Here, we analyzed if transdifferentiation-dependent induction of ECM components, e.g., collagen type I and thrombospondin-2 (TSP-2), and phenotypic changes are coregulated events and if both processes are mediated via TGF-beta pathway(s). Blocking the TGF-beta-dependent p38 MAPK pathway in HSC with the specific inhibitor SB203580 strongly reduces collagen I and TSP-2 mRNA expression without inhibiting upregulation of the typical MFB-marker, alpha-smooth-muscle actin (alpha-SMA). Similarly, interference with the Smad2/3/4 pathway using dexamethasone also heavily decreased expression of collagen type I and TSP-2 whereas transdifferentiation of HSC to the typical morphology of MFB with loss of fat droplets and increasing alpha-SMA was unchanged. Further, p38 MAPK mediated induction of collagen I and TSP-2 expression by TGF-beta1 was still achieved in the presence of dexamethasone, showing that dexamethasone does not block p38 while it delays Smad2 phosphorylation and antagonizes stimulation of a Smad3/Smad4 dependent TGF-beta reporter construct. Interestingly, in contrast to SB203580 and dexamethasone, overexpression of the TGF-beta antagonist Smad7 reduced ECM expression and simultaneously inhibited morphologic transdifferentiation, indicating that Smad7 fulfills additional features in HSC. In conclusion, our data show that phenotypic changes of transdifferentiating HSC and induction of matrix synthesis are independent processes, the latter being stimulated by both, Smad dependent and MAPK dependent TGF-beta signaling.

Actins↗

PubMed2004

Glucocorticoids decrease the bioavailability of TGF-beta which leads to a reduced TGF-beta signaling in hepatic stellate cells.

Glucocorticoids bound to their receptors transmit information, which regulates numerous physiological and pathophysiological responses, amongst others glucose metabolism, wound healing, inflammation, and stress, either directly as transcription factors by binding DNA elements of target genes or indirectly by protein-protein interactions with other transcription factors. TGF-beta, a key factor in activation of hepatic stellate cells (HSC), induces production of extracellular matrix, this being a prerequisite for the development of liver fibrosis. Glucocorticoids and their receptors may provide a crosstalk with the TGF-beta-Smad signaling pathway by antagonizing TGF-beta effects. We studied the influence of glucocorticoids on the TGF-beta isoform and Smad mRNA expression, TGF-beta secretion, and signaling in activated HSC using gene-specific real-time PCR, ELISA, and transfection techniques. Dexamethasone treatment reduces TGF-beta mRNA transcription in a time-dependent manner. Activated HSC produce TGF-beta and secrete it into the cell culture medium. After dexamethasone treatment, TGF-beta secretion into the medium is reduced dose-dependently but restorable by mifepristone. Further, we found that reduced secretion of endogenous TGF-beta is accompanied by a reduced TGF-beta signal. Additionally, reporter gene analysis after adenoviral infection with a recombinant virus encoding a Smad-binding-element showed that TGF-beta-Smad signaling is significantly down-regulated by dexamethasone in primary HSC and CFSC, a HSC related cell line. Our data suggest that glucocorticoids inhibit TGF-beta expression, prevent TGF-beta from efficient secretion, and finally lead to reduced TGF-beta signaling in primary HSC.

Adrenal Cortex Hormones↗

PubMed2004

Corticosteroids stimulate selectively transforming growth factor (TGF)-beta receptor type III expression in transdifferentiating hepatic stellate cells.

BACKGROUND/AIMS: Transforming growth factor (TGF)-beta receptors mediate TGF-beta signaling in activated hepatic stellate cells (HSC). This leads to pleiotropic cellular effects, e.g. to the production of extracellular matrix which is a hallmark for the development of liver fibrosis. Glucocorticoids and their receptors interact with the TGF-beta signaling pathway on the transcriptional and translational level. METHODS: To characterize TGF-beta receptor expression during HSC transdifferentiation and to study the influence of corticosteroids on receptor transcription in several liver cells, we established a real-time polymerase chain reaction procedure for mRNA quantification with gene-specific standards. RESULTS: All three TGF-beta receptor mRNAs are present in HSC and myofibroblasts. Whereas TGF beta receptor type I (T beta RI) shows a comparable mRNA expression during HSC transdifferentiation, T beta RII and T beta RIII mRNA concentration decreases in the course of time. In comparison with activated HSC T beta RIII mRNA is very low expressed in freshly isolated Kupffer cells and hepatocytes. Eight hours after corticosteroid treatment T beta RIII mRNA increased significantly in a time-and dose-dependent manner while the mRNA expression of T beta RI and T beta RII is not altered. The degree of induction of T beta RIII mRNA levels is also dependent upon the nature of the stimulating hormone: dexamethasone, hydrocortisone and aldosterone show different effects. CONCLUSIONS: The increase of T beta RIII by corticosteroids indicates that these hormones are important regulators of this receptor and thereby they can modulate TGF-beta signaling.

Activin Receptors, Type I↗