Paulsson, Mats / Wagener, Raimund / Sengle, Gerhard - C 7

Extracellular microfibrillar systems in disease pathogenesis: functional interactions in cytokine regulation, cellular differentiation and tissue homeostasis

Evidence from human disease and biochemical data clearly demonstrate that extracellular microfibrillar systems made of intricate networks of fibrillin, elastin/ EMILIN and collagen VI are controlling tissue integrity and cell fate. The aim of this project is to identify and characterize the functional interactions by which extracellular microfibrillar systems exert control over tissue homeostasis and how disturbances in these systems caused by mutations lead to disease.

Introduction

Tissue microenvironments formed by extracellular matrix (ECM) networks play an important role in regulating tissue structure and function. Extracellular microfibrillar networks (EMFN) made of fibrillin (fibrillin-1 and -2), elastin together with elastin microfibrillar interface proteins (EMILINs), and collagen VI with their associated ligands are of particular interest in this regard since they are ubiquitously found to surround cells in close proximity to basement membranes, and thereby guiding proper cellular phenotypes and functional behaviors of specialized cell types.

Human mutations in components of EMFN give similar phenotypic outcomes suggesting that EMFN work in concert to balance tissue homeostasis. Ulrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), congenital contractural arachnodayctyly (CCA) and Marfan syndrome (MFS) are congenital disorders characterized by features affecting the musculoskeletal and dermal system. Patients share distinct features like muscle weakness, contractures, kyphoscoliosis, extensible skin and the formation of spontaneous keloid scars. However, the underlying molecular mechanisms leading to these features remain largely obscure.

Fibrillin microfibrils are interconnected with other fibrillar networks important for growth factor regulation

We wanted to know whether the matrix incorporation of EMILINs (Elastin Microfibril Interface Located proteINs) which have been shown to be modulators of TGF-β and Wnt signalling pathways, depends on fibrillin microfibrils. Ultrastructural analysis of skin by EM after immunogold labeling, together with confocal immunofluorescence microscopy and size exclusion chromatography of skin extracts, demonstrated that EMILIN-1 and -2 in vivo do not form networks that depend on each other but are rather exclusively targeted to fibrillin-1 and -2 (Schiavinato et al., 2016).

Interestingly, in states of microfibrillar disease, such as in the murine GT-8 model of Marfan syndrome, characterized by progressive degradation of the fibrillin microfibril network, we found that both EMILIN networks are affected (Schiavinato et al., 2016) (Figure 1A). This indicates that EMILIN-1 and -2 alterations may play a role in the pathomechanisms of the fibrillinopathies, where microfibril destabilization due to fibrillin-1 or -2 deficiency leads to multisystemic features characterized by reduced tissue integrity and global activation of growth factor signaling.

Fibrillin microfibrils are integration platforms for BMP signaling

We found that genetic ablation of Fbn2 resulted in increased BMP signalling in skeletal muscle which resulted in a dystrophic phenotype indicated by reduced muscle mass and abnormal fat deposition (Sengle et al., 2015) (Figure 1B).

Muscle mass and fat deposition was rescued by additional ablation of one Bmp7 allele corroborating that increased BMP activity was causative for this phenotype (Sengle et al., 2015). Moreover, our biochemical investigations also showed that direct binding of BMPs to fibrillin-1 changes their activation state from bioactive to latent (Wohl et al., 2016).

Using electron microscopy, small angle X-ray scattering, and circular dichroism spectroscopy, we could demonstrate that the BMP-7 prodomain-growth factor complex assumes an open V-like structure when it is bioactive, however, upon binding to fibrillin-1 it is rendered into a closed ring-shape which denies access of BMP receptors to the growth factor (Wohl et al., 2016).

Structural insights into collagen VI microfibrils

We crystallized the N5 domain of the collagen VI α3 chain as a prototype domain and solved the structure at 1.2 Å resolution (Becker et al., 2014) (Figure 1 C). To get insight into the pathogenesis of collagen VI myopathies we also crystallized the N5 domain carrying a Ullrich congentital muscular dystrophy (UCMD)-causing point mutation.

We used the structure of the N5 domain for homology modeling and were thereby able to locate the exact spatial position of a number of disease-associated mutations and on this basis we can now analyze their structural impact. This will allow a mechanistic analysis of collagen-VI-associated muscular dystrophy phenotypes. Furthermore, structural features of the three novel long collagen VI chains (α4, α5, and α6) and the assembly of these into tetramers and microfibrils were investigated (Maaß et al., 2016). N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the chains showed a C-shaped structure similar to the α3 chain. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble into homotetramers that are incorporated into mixed microfibrils (Maaß et al., 2016) (Figure 1D).

Perspectives

Our findings corroborate the emerging concept that extracellular matrix proteins are actively regulating growth factor activity and bioavailability. This concept opens novel treatment avenues for a wide range of connective tissue diseases which were thought to be incurable due to defects in proteins of purely architectural nature.

Selected publications

Maaß, T., Bayley, C.P., Mörgelin, M., Lettmann, S., Bonaldo, P., Paulsson, M., Baldock, C.*, and Wagener, R.* (2016). Heterogeneity of collagen VI microfibrils: Structural analysis of non-collagenous regions, J. Biol. Chem. 291, 5247-5258. *equal contribution.

Wohl, A.P., Troilo, H., Collins, R.F., Baldock, C., Sengle, G. (2016). Extracellular regulation of bone morphogenetic protein activity by the microfibril component fibrillin-1. J. Biol. Chem. 291, 12732-46.

Schiavinato, A., Keene, D.R., Wohl, A.P., Corallo, D., Colombatti, A., Wagener, R., Paulsson, M., Bonaldo, P.*, Sengle, G.* (2016). Targeting of EMILIN-1 and EMILIN-2 to fibrillin microfibrils facilitates their incorporation into the extracellular matrix. J. Invest. Dermatol. 136, 1150-1160. * equal contribution.

Becker, A.K.§, Mikolajek, H. §, Paulsson, M., and Wagener, R.*, Werner, J.M.* (2014) A novel structure of a collagen VI VWA-domain displays N- and C-termini at opposite sides of the protein. Structure 22, 199-208. § and * equal contribution.

Sengle, G., Carlberg, V., Tufa, S.F., Charbonneau, N.L., Smaldone, S., Carlson, E.J., Ramirez, F., Keene, D.R., Sakai, L.Y. (2015). Abnormal activation of BMP signaling causes myopathy in Fbn2 null mice. PLoS Genet. 11, e1005340.

Former Funding Period 01/2014 - 12/2016

Information from this funding period will not be updated anymore. New research related information is available here.


Prof. Dr. rer. nat. Mats Paulsson

Institute for Biochemistry II

Prof. Dr. rer. nat. Mats Paulsson

Co-Principal Investigator C 14
Executive Board Member

mats.paulsson@uni-koeln.de

Work +49 221 478 6997

Fax (Work) +49 221 478 6977

Institute for Biochemistry II
Joseph-Stelzmann-Str. 52
50931 Cologne

http://www.uni-koeln.de/med-fak/biochemie/biomed2/

Publications - Mats Paulsson

Link to PubMed


Prof. Dr. rer. nat. Raimund Wagener

Institute for Biochemistry II

Prof. Dr. rer. nat. Raimund Wagener

Co-Principal Investigator C 14

raimund.wagener@uni-koeln.de

Work +49 221 478 6990

Fax (Work) +49 221 478 6977

Institute for Biochemistry II
Joseph-Stelzmann-Str. 52
50931 Cologne

http://www.uni-koeln.de/med-fak/biochemie/staff/wagener/

Publications - Raimud Wagener

Link to PubMed


PD Dr. rer. nat. Gerhard Sengle

Institute for Biochemistry II

PD Dr. rer. nat. Gerhard Sengle

Principal Investigator C 14

gsengle@uni-koeln.de

Work +49 221 478 97260

Fax (Work) +49 221 478 6977

Institute for Biochemistry II
Joseph-Stelzmann-Str. 52
50931 Cologne

http://www.uni-koeln.de/med-fak/biochemie/staff/sengle/

Publications - Gerhard Sengle

Link to PubMed

Affiliations

CRC 829

CECAD Cologne

Group Members

Ann-Kathrin Becker (former PhD student)
Stefanie Heumüller (PhD student)
Katrin Hildebrandt (PhD student)
Tobias Maaß (former PhD student)
Alvise Schiavinato (guest scientist)
Alexander Wohl (former PhD student)

Figure 1

Fig. 1: (A) Fibrillin fibers target EMILIN-1 and are progressively affected in skin of a Marfan mouse model.
(B) Fibrillin-2 ablation leads to reduced muscle mass and aberrant BMP signalling in forelimb muscle.
(C) Structure of Col6a3N5.
(D) Immuno EM of mixed collagen VI microfibrils using gold-labelled antibodies against the alpha3 (small particles) and the α5 chain (large particles) (left) and schematic drawing illustrating the microfibril composition (right).