Epigenetics and Gene Expression
Tissue Engineering
Injury to the articular surface, when left untreated, can lead to early onset of osteoarthritis. Repair technology such as Autologous Chondrocyte Transplantation (ACT) is among the first examples of Tissue Engineering (TE) but encounters several drawbacks, i.a. variability in tissue quality, costs and complex logistics. In both ACT and Autologous osteochondral grafting, cartilage is harvested from the joint and thus adverse effects to joint homeostasis must be considered. We recently developed a novel method to generate chondrogenic tissue in vivo: the in situ incubator (ISI). ISI involves creating a space between bone and periosteum, this space is filled by a biogel to generate extra-articular cartilage and thereby differs radically from current Tissue Engineering techniques. In preliminary studies, we found that by simply altering the environment of the ISI, e.g. by induction of hypoxia by local delivery of Suramin, cartilage generation is favored (Fig. 1). An ex vivo model has been optimized in which a whole graft of undifferentiated periosteum is embedded in agarose and stimulated to chondrogenesis. This model enables us to study environmental factors (growth factors, cytokines, oxygen and pharmaceutics etc.) needed to further evaluate this novel technology of de novo cartilage formation using the ISI and to shed light on what biogel characteristics favorably direct the ISI toward chondrogenesis. The overall aim is to improve our understanding what conditions influence succesrate, quality and quantity of cartilage generated in the ISI and to uncover the full therapeutic potential of the "in situ incubator" concept for tissue engineering of cartilage.
Figure:
(A) Extra-articular cartilage (ec) located distally from the rabbit knee (k).
(B) The total amount of extra-articular cartilage formed is approx. 1 cm in diameter. Note 2 grafts of 3 mm in diameter were cored out.
(C-F) Safarin-O stained sections of tissue formed in the In Situ Incubator.
(C) Cartilage formation after injection with HA + liposomes.
(D) Aspecific tissue reaction after injection with agarose/PRP. Note that no cartilage is formed
(E) Cartilage formation after injection with agarose only. The gap indicated by * is probably caused during harvest of the extra-articular cartilage.
(F) Is an enlargement of the box in (E).
Original magnification (C) 25X; (D) 100X; (E) 50X; (F) 200X
Research group
Dr. L.W. van Rhijn, projectleader
Dr. J.W. Voncken
Prof.Dr. R.G.T. Geesink
P. Deckers
N. Guldemond
V.P. Shastri (Vanderbilt, Nashville, TN)
Post-doctoral fellows
Dr. T.J.M. Welting
PhD Students
E.J.P. Jansen
P.J. Emans
F. Spaapen
Technicians
D.A.M. Surtel
A. Cremers
G. M. Wetzels
Students
N. Wijnands
D. v Iersel
Molecular Epigenetics
Each cell carries both genetic and epigenetic information in its nucleus. DNA is not naked, but is tightly packed in chromatin inside a eukaryotic nucleus. Epigenetic processes control the use of genetic information (i.e. gene-expression) by changing chromatin structure. This is what underlies cell diversity in the human body: whereas virtually all cells have the same DNA, each cell uses different genes. In genetically identical monozygotic twins, non-identical epigenetic information contributes to differences in susceptibility to disease. In addition, there are strong indications that epigenetic information can be inherited from one to the next generation (cells, organisms). The environment (i.a. food, lifestyle) is gaining importance as far as being able to influence epigenetic regulatory mechanisms and thereby gene expression is concerned. However, fairly little is known about the exact molecular mechanisms underlying these processes, which are relevant for human development en disease.
Our group investigates signaling to chromatin. The main focus of our research is to understand how epigenetic control of chromatin structure by Polycomb Group (PcG) proteins is regulated. PcG install a transcriptional memory in chromatin structure; they do this by writing and reading covalent post-translational modifications on histone tails, which collectively act as an epigenetic register. We recently described a novel link between cell signaling and chromatin modifiers: Polycomb Group proteins (PcG) are phosphorylated in vivo by MAPKAP kinases; PcG-phosphorylation correlates with chromatin dissociation. Relevantly, as MAPKAPKs are down-stream mediators of canonical MAPK signaling cascades, our findings suggest a novel mechanism used by cells to dynamically alter gene expression in response to environmental signals.
Virtually nothing is known about regulation of PcG-function and its implications for chromatin structure modification. Our research proposal focuses on revealing the biological relevance (why do cells need it? fig. A) and the molecular epigenetics (what happens at the chromatin level; fig. B) of MAPKAPk-to-PcG signaling and PcG-phosphorylation (fig. C). Our models focus on proliferation, differentiation, senescence and cell stress, all processes which are implicated in normal and abnormal growth. Our most recent discoveries suggest crucial roles for MAPKAPKs and PcG, as loss of either of these two proteins classes in established cell lines results in cell cycle exit (established cell lines) or defective differentiation, in addition to abnormal expansion (progenitor/stem cells).
Figure: Molecular Epigenetics. A) MAPKAPK3 associates with PcG targets on chromatin (chromatin immunoprecipitation). B) Loss-of-function models for PcG (shown) or MAPKAPK3 interferes with differentiation (osteo/chondrogenesis). C) MAPKAPK3 phosphorylates PcG proteins in vitro (peptide spot arrays; CREB, HSP27: positive controls)
Research group
J.W. Voncken, project leader
PhD students
H. Niessen
F. Spaapen
C. Rofel
E. Poliard
N. Kubben
E. Bardina
Technical support
V. Dahlmans van Leeuwen
C. Geijselaers
R. Hessing
Undergraduates
J. Eygelshoven
P. Cornelissen
S. Bartels
C. Munts
M. Haagmans