Gerhard Sengle / Raimund Wagener / Mats Paulsson - C 14

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

New strategies for treating congenital musculoskeletal disorders are needed. Current treatments are limited and aim to prolong ambulation and survival. Cellular microenvironments such as stem cell niches in muscle and bone are defined by extracellular microfibrillar networks (EMFN) which are required for tissue structure and function. EMFN made of fibrillin-1 and -2, and collagen VI with associated ligands are of particular interest since human mutations in EMFN components result in disorders with overlapping clinical features.

Introduction

The molecular mechanisms by which perturbed cell-matrix interactions lead to musculoskeletal disorders are not well understood, and common or shared pathogenetic mechanisms have not yet been uncovered. From genetic evidence in humans and mice it becomes clear that EMFN together with their associated ligands are controlling homeostasis of multiple tissues. However, the underlying molecular pathways remain obscure. Our previous investigations have identified EMFN consisting of fibrillins as regulators/ modulators of growth factor activity. This corroborates 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.

EMILIN-1 is required for extracellular matrix deposition of fibulin-4

EMILINs (Elastin-Microfibril-Interface-Located-proteINs) comprise a family of three structurally homologous extracellular matrix (ECM) glycoproteins that serve as versatile regulators of cell adhesion, migration, and proliferation, but also as unique modulators of extracellular signalling pathways. EMILINs were found to influence pro-TGF-β processing, modulate Wnt and Hedgehog signaling, activate death receptor mediated apoptosis, and communicate with cells via α4β1 and α9β1 integrin mediated signalling. We recently found that EMILINs are targeted to fibrillin microfibrils implicating them in the disease pathogenesis of fibrillin-related disorders (Schiavinato et al., 2016). Recently, we showed that EMILIN-1 directly interacts with fibulin-4 and is required for its ECM deposition within specialized cellular microenvironments (Schiavinato et al., 2017). Fibulin-4 mutations lead to flexion contractures in patients and mice. Mutations in fibrillin-2 also result in flexion contractures of the large joints in Beals Syndrome patients probably due to dysregulated BMP signalling causing a delay in muscle maturation as previously shown in fibrillin-2 null mice (Sengle et al. 2015).

Assessment of structurally impaired fibrillin microfibrils by Raman microspectroscopy in a mouse model of Marfan Syndrome

Mutations in building blocks of the EMFN manifest in connective tissue disorders such as Marfan syndrome (MFS) characterized by long bone overgrowth, aortic aneurysm formation, and muscle weakness. Understanding how structural changes induced by fibrillin-1 mutation impact the architecture of fibrillin microfibrils (FMF), which then translates into an altered activation state of targeted growth factors, represents a huge challenge in elucidating the genotype-phenotype correlations in MFS which is caused by over 3000 known FBN1 mutations. In collaboration with the group of Katja Schenke-Layland (University of Tübingen) we could show that Raman microspectroscopy is able to reveal structural changes in fibrillin-1 microfibrils and elastic fiber networks and to discriminate between normal and diseased networks in vivo and in vitro (Brauchle et al., 2017). For this purpose we analyzed a Marfan mouse model in which the C-terminal half of fibrillin-1 is truncated. Skin biopsies and organotypic co-cultures from isolated primary fibroblasts were utilized for Raman measurements. In our study, structural elements in an area of 400 × 700 nm were scanned based on the volume of the Raman laser spot. Given the length of fibrillin-1 monomers of 150 nm and a 50-80 nm periodicity of the “beads-on-a-string”-like ultrastructure where eight monomers convene at each bead, Raman microspectroscopy is able to pick up structural changes of FMF on the molecular level. Our data convincingly showed that Raman microspectroscopy is indeed sensitive enough to detect molecular differences between wild-type and mutant fibrillin-1 fibers. We detected that C-terminally truncated fibrillin-1 resulted in fibrillin fibers that contain fewer signals from CH-deformations (1450 cm−1) and amide I (1668 cm−1), whereas the amide III signal (1245 cm−1) was increased in destabilized mutant fibrillin-1 fibers. Therefore Raman microspectroscopy may be utilized as a non-invasive and sensitive diagnostic tool to identify connective tissue disorders and monitor their disease progression.

Collagen VI chains play a role in tendon repair

Collagen VI is a microfibrillar collagen which when mutated gives rise to disorders such as Bethlem myopathy (BM) or Ullrich congenital muscular dystrophy (UCMD). Recently, we found a potential regulatory role of the newly identified collagen VI chains in tendon repair mechanism (Sabatelli et al., 2016). We studied the expression of collagen VI α5 and α6 chains in normal human tendon fibroblast cultures, both under basal condition and in response to TGF-β1, a potent regulator of tendon healing. Under basal conditions, the α5 chain was expressed to a lesser extent compared to the α3 chain, while the α6 chain was absent. TGF-β1 treatment induced an opposite effect on α5 and α6 chain expression. While the α5 chain was dramatically reduced, the α6 chain was induced and released into the culture medium. Our data indicate that collagen VI α5 and α6 chains are differentially involved in tendon matrix homeostasis. The α6 chain may represent a new potential biomarker for monitoring TGF-β1-related events in tendon, as healing and fibrotic scar formation.

Perspectives

This project has the potential to provide a comprehensive and fundamental understanding of the molecular pathomechanisms caused by mutations in EMFN components which will lay the foundation for future translational approaches of congenital musculoskeletal disorders

Selected publications

Brauchle, E., Bauer, H., Fernes, P., Zuk, A., Schenke-Layland, K., and Sengle, G. (2017). Raman microspectroscopy as a diagnostic tool for the non-invasive analysis of fibrillin-1 deficiency in the skin and in the in vitro skin models. Acta Biomater. 52: 41-48.

Sabatelli, P., Sardone, F., Traina F3, Merlini L4, Santi S1,2, Wagener, R., and Faldini, C (2016). TGF-β1 differentially modulates the collagen VI α5 and α6 chains in human tendon cultures. J Biol Regul Homeost Agents. 2016, 30:107-113.

Sengle, G., Carlberg, V., Tufa, S.F., Charbonneau, N.L., Smaldone, S., Carlson, E.J., Ramirez, F., Keene, D.R., and Sakai, L.Y. (2015). Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice. PLoS Genet. 11: e1005340.

Schiavinato, A., Keene, D.R., Wohl, A.P., Corallo, D., Colombatti, A., Wagener, R., Paulsson, M., Bonaldo, P., and Sengle, G. (2016). Targeting of EMILIN-1 and EMILIN-2 to Fibrillin Microfibrils Facilitates their Incorporation into the Extracellular Matrix. J Invest Dermatol. 136:1150-60.

Schiavinato, A., Keene, D.R., Imhof, T., Doliana, R., Sasaki, T., and Sengle G. (2017). Fibulin-4 deposition requires EMILIN-1 in the extracellular matrix of osteoblasts. Sci Rep. 7: 5526.


PD Dr. Gerhard Sengle

Institute for Biochemistry II

PD Dr. 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


Prof. Dr. Raimund Wagener

Institute for Biochemistry II

Prof. Dr. 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 - Raimund Wagener

Link to PubMed


Prof. Dr. Mats Paulsson

Institute for Biochemistry II

Prof. Dr. 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

Group Members

Katrin Hildebrandt (doctoral student)
Stefanie Heumüller (doctoral student)

Figure 1

Localization of EMFN in specialized cellular microenvironments. Top panel: Confocal immunofluorescence microscopy showing EMILIN-1 and -2, together with fibrillin-2 localization in transverse sections from newborn mouse tail. Each protein showed a specific distribution pattern among the different tissues present in the tail. The inset in the merged panel shows a higher magnification of the annulus fibrosus, where EMILIN-1 and fibrillin-2 fibers show co-localization. Bottom panel: siRNA mediated knock-down of EMILIN-1 abolished ECM deposition of fibulin-4 in MC3T3-E1 osteoblasts. Scale bar: 75 μm.