There is growing evidence that aminobisphosphonates like ibandronate show anticancer activity by an unknown mechanism. Biochemically, they prevent posttranslational isoprenylation of small GTPases, thus inhibiting their activity. In tumor cells, activated RAS-GTPase, the founding member of the gene family, down-regulates the expression of the pro-apoptotic gene FAS via epigenetic DNA-methylation by DNMT1. We compared ibandronate treatment in neoplastic human U-2 osteosarcoma and in mouse CCL-51 breast cancer cells as well as in the immortalized non-neoplastic MC3T3-E1 osteoblastic cells. Ibandronate attenuated cell proliferation in all cell lines tested. In the neoplastic cells we found up-regulation of caspases suggesting apoptosis. Further we found stimulation of FAS-expression as a result of epigenetic DNA demethylation that was due to down-regulation of DNMT1, which was rescued by re-isoprenylation by both geranylgeranyl-pyrophosphate and farnesylpyrophosphate. In contrast, ibandronate did not affect FAS and DNMT1 expression in MC3T3-E1 non-neoplastic cells. Data suggest that bisphosphonates via modulation of the activity of small-GTPases induce apoptosis in neoplastic cells by DNA-CpG-demethylation and stimulation of FAS-expression. In conclusion the shown epigenetic mechanism underlying the anti-neoplastic activity of farnesyl-transferase-inhibition, also explains the clinical success of other drugs, which target this pathway.

The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2−/−) mouse embryos.The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2−/− MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays.Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2−/− MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2−/− MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2.Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.

Thyroid hormones (T3,T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links. T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.

Mospd1 codes for a small protein with unknown physiological function, which is part of a family of genes, including Mospd2 and Mospd3, defined by the presence of the major sperm protein domain and two transmembrane domains. This work characterizes the Mospd1 gene, the intracellular location of the protein and its expression in different mouse tissues and mesenchymal cell lines during differentiation. The role of Mospd1 in mesenchymal cellular differentiation was studied by siRNA knockdown experiments in mouse osteoblastic MC3T3‐E1 cells. Transfection experiments of the targeted cDNA show MOSPD1 located in the endoplasmatic reticulum and in the Golgi apparatus. Removal of the last exon of the gene resulted in localization of the protein in the nucleus, which was attributed to a nuclear export sequence in the N‐terminal part. In mouse tissues the gene was generally strongly expressed while mesenchymal tissues showed the highest expression. In mesenchymal cell lines Mospd1 mRNA was higher expressed in cells with advanced differentiation status. In osteoblastic, myoblastic, and adipocytic cell lines Mospd1 was up‐regulated during differentiation. Genome‐wide gene expression analysis after knockdown of Mospd1 by siRNA in MC3T3‐E1 cells revealed a shift in the gene expression pattern from mesenchymal to epithelial genes featuring up‐regulation of the epithelial cadherin Cdh1 and down‐regulation of its inhibitors Snail1 and 2 and the mesenchymal cadherin Cdh11, suggesting a mesenchymal to epithelial transition. From these data we conclude that Mospd1 plays a pivotal role in the developmental regulation at the switch between mesenchymal and epithelial cells. J. Cell. Physiol. 226: 2505–2515, 2011. © 2010 Wiley‐Liss, Inc.

Epistasis is generally defined as the interaction between two or more genes or their mRNA or protein products to influence a single trait. Experimental evidence suggested that epistasis could be important in the determination of the genetic architecture of complex traits in domestic animals. Acetyl-coenzyme A carboxylase alpha (ACACA) and fatty acid binding protein 2 (FABP2) are both key factors of lipogenesis and transport. They may play a crucial role in the weight variability of abdominal adipose tissue in the growing chicken. In this study, the polymorphisms of c.2292G>A in ACACA and c.-561A>C in FABP2 were detected among individuals from two broiler lines which were divergently selected for abdominal fat content. Epistasis between the two SNPs on abdominal fat weight (AFW) and abdominal fat percentage (AFP) was analyzed. The additive × additive epistatic components between these two SNPs were found significant or suggestively significant on both AFW and AFP in lean lines of the 9th and 10th generation; whereas, it was not significantly associated with either AFW or AFP in fat lines. At the same time, there were not any other significant epistatic components found in both generations or in both lines. Significant epistatic effects between these two SNPs found only in the lean lines could partly be due to the fact that the abdominal fat traits in these two experimental lines have been greatly modified by strong artificial selection. The results suggested that the epistasis mode may be different between the lean and fat chicken lines. Our results could be helpful in further understanding the genetic interaction between candidate genes contributing to phenotypic variation of abdominal fat content in broilers.

Kollagen Typ I, der Hauptbestandteil der extrazellularen Matrix des Knochens, weist eine super-molekulare Organisation auf. Die einzelnen Kollagenmolekule werden einer Reihe von intra- und extrazellularen Modifikationen unterzogen, die es ihnen ermoglicht extrazellular Kollagenfibrillen auszubilden. Ein wesentlicher Schritt dabei ist die Ausbildung von Kollagenquervernetzungen. Dieser Prozess ist gewebespezifisch und wird von vielen zellularen und matrix-abhangigen Signalen gesteuert. Hemmung der Lysyloxidase (Lox), ein Schlusselenzym der Kollagenvernetzung, fuhrt zu veranderten Quervernetzungen und gestorter Fibrillogenese. Ergebnisse aus in-vitro als auch in-vivo durchgefuhrten Experimenten bestatigten, dass solche Veranderungen zu Knochenmineralverlust, reduzierter Knochenfestigkeit und einer veranderten Mineralisation fuhren. Ein weiterer Faktor der eine wesentliche Rolle in der Organ und Gewebsentwicklung spielt ist die Regulierung der Expression von Genen durch epigenetische DNA-Methylierung. Bis zum jetzigen Zeitpunkt ist die Bedeutung von diesem Mechanismus in der Knochenentwicklung und Pathogenese nur wenig erforscht worden. In dieser Dissertation wurde daher auf die Rolle epigenetischer Genregulationen im Osteoblasten intensiv eingegangen. Nach Behandlung der pre-osteoblastaren MC3T3-E1 Maus Zelllinie mit den zwei Inhibitoren der Lox, beta aminopropionitrile (bAPN) und Homocysteine (hcys), haben wir den Effekt von diesen zwei Lathyrogenen auf die Zelllinie analysiert und verglichen. Die Expression von osteoblastaren Genen wurde mittels „real time polymerase chain reaction“ (qPCR) und „gene expression microarrays“ untersucht. Marker der osteoblastischen Aktivitat und Zellproliferation wurden durch Messung der Aktivitat der Alkalischen Phosphatase sowie Viabilitatstest bestimmt. Die Effekte der Substanzen auf die Kollagenquervernetzung wurden durch „Fourier-Transform-Infrarot-Spektrometrie“ (FTIR) gemessen. Die ersten Ergebnisse zeigten dass sowohl bAPN als auch hcys Lox nicht nur enzymatisch hemmen sondern auch dessen mRNA Expression vermindern. Unter Berucksichtigung der klinischen Bedeutung von hcys, haben wir den zellularen Signalweg fur die hcys-abhangige Verminderung der Lox Expression erforscht. Dabei nutzten wir Techniken wie „Enzyme-linked immunosorbent assay“ (ELISA), Immuno Blotting und Chromatin-Immunoprazipitation (ChIP). Auswertungen ergaben das Interleukin 6 (IL-6), der Transkriptionsfaktor „Friend leukemia integration 1“ (FLI1) und die DNA-Methyltransferse 1 (DNMT1) in der Repression von Lox durch hcys involviert sind. Untersuchungen an der Promotorregion des Lox-Gens ergaben eine DNA-methylierungsabhangige Regulation der Lox Expression durch hcys. Im letzten Teil der Arbeit wurde ein neuer Signal–Transduktionsweg aufgeklart, durch den die extrazellulare Matrix (ECM) die Proliferation und Differenzierung von Osteoblasten fordern kann. Beim Aussahen von MC3T3-E1 Zellen auf mit Kollagen Typ I beschichtete Platten haben wir eine erhohte Expression an osteoblastaren Genen und eine verminderte Expression des pro-apoptotischen Gens Fas beobachtet. Durch die Verwendung verschiedener Inhibitoren, qPCR und DNA-Methylierungsanalysen konnten wir zeigen, dass das extrazellulare Kollagen Typ I via FAK, MAPK und den Transkriptionsfaktor AP1 direkt die Expression des Gens Dnmt1 stimuliert was in weiterer Folge fur die Stilllegung des Gens Fas durch epigenetischer DNA-Methylierung verantwortlich ist.

It is well known that suramin influences proliferation and differentiation of tumour cells. To study whether and how suramin effects osteosarcoma (OS) cells, proliferation, differentiation, LOX mRNA expression and telomerase activity (TA) was analysed in the human MG-63 and U-2 OS, and the rat UMR-106 OS cell lines. Data show that suramin inhibited proliferation in the human cell lines and upregulated alkaline phosphatase activity. TA was attenuated in the human cells while in UMR-106 it was not changed. In UMR-106 suramin had no influence on osteocalcin and LOX expression, in the human cells however, both genes were upregulated.

Osteoblasts synthesize collagen matrix, which itself regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase (LOX), which is involved in the collagen cross-linking process. Lathyrogens, like ß-aminopropionitrile (ßAPN), inhibit the formation of a stable matrix. The aim of the present study was to investigate the influence of cross-linking on osteoblastic differentiation. MC3T3-E1 cells were seeded and treated with or without 400 μM ßAPN for 1 week. Thereafter, living cells were removed and, on this extracellular matrix, new MC3T3-E1 cells were seeded and cultured for 1 week without ßAPN. RNA was isolated, and expression of specific marker genes was determined by quantitative reverse transcription-polymerase chain reaction. Changes in specific cross-links after ßAPN treatment were measured with Fourier-transform infrared spectroscopy. The collagen matrix that formed showed a significant reduction of two major cross-links of bone collagen, deH-DHLNL and pyr, compared to control cultures. Gene expression studies showed an increase of collagen α1 (I) (COL1A1) to 150%. Expression of LOX and osteocalcin (OCN) mRNA was significantly downregulated to about 75%. When fresh MC3T3-E1 cells were seeded on this altered matrix without ßAPN, COL1A1 mRNA expression was upregulated (140%), OCN was downregulated (60%), and LOX mRNA expression remained unaffected. These results indicate that ßAPN treatment not only disrupts collagen cross-link formation but also affects osteoblastic activity and expression. In conclusion, the disrupted matrix produced in the presence of lathyrogen influences, even in its absence, the expression of osteoblastic genes.