Project Leader: Prof. Taina Pihlajaniemi, M.D., Ph.D.
Oulu Center for Cell-Matrix Research, Department of Medical Biochemistry and Molecular Biology, Institute of Biomedicine, Faculty of Medicine, University of Oulu
 

Background and Significance

Cells receive survival and positional information from the ECM and it is now evident that ECM biology permeates all aspects of cellular function. Moreover, the ECM is associated with a wide spectrum of common human diseases including arthritis, atherosclerosis, cancer, diabetes, fibrosis, skeletal deformity and poor wound healing.

Twenty-nine types of collagen have been found in vertebrates, and in this project we address the significance of two subgroups of collagens, the basement membrane (BM)-associated collagens XV and XVIII, and the subgroup of collagens XIII, XXIII and XXV. Collagens of the latter subgroup occur both as transmembrane and shed forms, and they may have not only structural but also important regulatory functions. Collagen XVIII is of considerable interest because of its C-terminal endostatin domain, which has been shown to inhibit angiogenesis and tumour formation.

Collagen XIII has a short cytosolic domain and a largely triple-helical ectodomain. As a result of its location at many cell–matrix junctions and in focal adhesions in cultured cells it conceivably has a function in cell adhesive processes. Human disorders arising from mutations in this collagen have not been identified, but our work with genetically modified mice suggests functional roles for collagen XIII at least in bone, muscle and the placenta. Collagens XV and XVIII are multidomain proteins found in association with most BMs. Mutations in collagen XVIII lead to Knobloch syndrome, characterised by myopia and occipital encephalocele. Although single and double knockout mice as regards collagens XV and XVIII are viable and fertile, the lack of these related collagens affects the integrity of several organs and tissues such as eyes, brain ventricles, skeletal muscle, heart and blood vessels. Both the transmembrane and endostatin collagens are associated with malignant processes.

Alterations in the vasculature are involved in many common human diseases, such as malignant tumours and cardiac disorders, and mutations in genes important for blood vessel morphogenesis cause vascular anomalies. Angiopoietin growth factors (Ang1–4) and Tie receptors (Tie1 and Tie2) are important for vascular morphogenesis and regulate vessel integrity in cooperation with perivascular ECM.

Recent Progress

The physiological significance of collagen XIII is being addressed by using novel gene-targeted and transgenic mouse lines. Earlier work with transgenic mice overexpressing mutant collagen XIII has shown that this collagen affects the normal development and function of the foetal heart and placenta, and that it is involved in the maintenance of the intestinal BM, where it regulates immune responses and cancer susceptibility. Our recently generated null and knock-in reporter mice have made it possible to identify tissues susceptible to lack of collagen XIII. The first results indicate that collagen XIII is a novel muscle-derived regulator of motor synapse differentiation and stability. It affects maturation of acetylcholine receptor clusters, apparently in an autocrine manner via its shed ectodomain, and trans-synaptic adhesion. Altogether, when collagen XIII is lacking, the structure of neuromuscular junctions is severely affected and neurotransmission is compromised.

Lack of collagen XV in knock-out mice (Col15a1 ^-/-) leads to a mild, progressive myopathy and morphological alterations in capillaries. More detailed study on the in vivo functions of collagen XV highlights the importance of this collagen in the heart. Collagen XV deficiency was found to lead to a complex cardiac phenotype and to predispose the null mice to pathological responses under cardiac stress. The structure and function of microvessels was compromised in the null mice. Moreover, collagen XV was found to contribute to peripheral nerve maturation and C-fibre formation. The latter work provides the first in vivo evidence of collagens playing important physiological roles in axonal segregation and myelination by Schwann cells. The mechanisms whereby collagen XV exerts its functions are suggested by ultrastructural findings, which point to a role of collagen XV in organising the fibrillar collagen matrix in the heart and nerves. Moreover, recombinant collagen XV was found to modify the fibronectin matrix and its cell-binding properties.

Collagen XVIII occurs in three N-terminal isoforms, the “short form” being derived from transcription of promoter 1, and the long and middle forms being derived from promoter 2 and alternative splicing of exon 3 sequences. We have recently generated mutant mice specifically lacking either promoter 1- or 2-derived transcription, allowing studies on the roles of the promoter-specific collagen XVIII isoforms in tissues. Characterisation of kidneys of the mutant mice showed that lack of the long and middle forms leads to glomerular podocyte effacement, while lack of the short form alters the BM of proximal tubules. These abnormalities lead to mild disparate effects on kidney function with respect to creatinine filtration.

Tumour growth and metastasis are dependent on lymphangiogenesis and angiogenesis and their inhibition is a valuable approach to cancer therapy. Up-regulation of the Tie2 ligand, Ang2, is one of the first markers of tumour vasculature, and high concentrations of Ang2 have been associated with many human cancers. To understand better the mechanisms whereby Ang2 may contribute to an angiogenic phenotype we have studied Ang2-regulated Tie2 trafficking, activation and cellular effects in cultured cells and in mouse models in collaboration with Kari Alitalo’s and Pipsa Saharinen’s groups (University of Helsinki). We found that Ang2-specific Tie2 trafficking to cell–matrix contacts may contribute to the destabilisation of vasculature when angiogenesis is initiated, while Ang2 blocking increased vascular integrity and decreased metastatic dissemination.

Moreover, in work carried out in collaboration with Veli-Matti Kähäri’s group (University of Turku) using human tissue samples, cutaneous squamous cell carcinoma (SCC) cell lines and chemically induced mouse skin SCCs, SerpinA1 was identified as a novel tumour cell-associated biomarker of progression of cutaneous SCCs. In a study with Kari Alitalo’s group, deletion of the endothelial Bmx tyrosine kinase was found to impair tumour angiogenesis and growth in mice.

Future Goals

We will test our hypothesis that members of the transmembrane and endostatin collagen families are complex multidomain proteins with not only structural functions but also important regulatory roles in differentiation, organogenesis, inflammation and tumorigenesis. The medical significance of cell–ECM homeostasis will be addressed in terms of target identification and validation in order to develop new diagnostic, prognostic and therapeutic measures. Part of our work is associated with that of the Finnish Institute of Molecular Medicine, University of Helsinki, and this association is aimed at facilitating the translational aspects of our project. The main objective of Dr. Lauri Eklund is to gain an understanding at the molecular level of the mechanisms that mediate and regulate Tie2 trafficking and Tie2 functions in subcellular domains, with special focus on endothelial cell–ECM interactions. Another goal is to better characterize the altered function of Tie2 resulting from mutations responsible for familial and sporadic venous malformations.

Publications 2011-

Kinnunen AI, Sormunen R, Elamaa H, Seppinen L, Miller RT, Ninomiya Y, Janmey PA, Pihlajaniemi T. Lack of collagen XVIII long isoforms affects kidney podocytes, whereas the short form is needed in the proximal tubular basement membrane. J Biol Chem 286:7755-7764, 2011.

Farshchian M, Kivisaari A, Ala-aho R, Riihilä P, Kallajoki M, Grénman R, Peltonen J, Pihlajaniemi T, Heljasvaara R, Kähäri V-M. Serpin peptidase inhibitor clade A member 1 (SerpinA1) is a novel biomarker for progression of cutaneous squamous cell carcinoma. Am J Pathol 179:1110-1119, 2011.

Seppinen L and Pihlajaniemi T. The multiple functions of collagen XVIII in development and disease. Matrix Biol 30:83-92, 2011.

VEGF and angiopoietin signaling in tumor angiogenesis and metastasis. Saharinen P, Eklund L, Pulkki K, Bono P, Alitalo K. Trends Mol Med 17:347-362, 2011.

Holopainen T, López-Alpuche V*, Zheng W*, Heljasvaara R, Tvorogov D, D’Amico G, Pihlajaniemi T, Alitalo K. Deletion of the endothelial Bmx tyrosine kinase impairs tumor angiogenesis and growth. *Equal contribution. Cancer Res, in press.

Holopainen T, Saharinen P, D’Amico G, Lampinen A, Eklund L, Sormunen R, Anisimov A, Zarkada G, Lohela M, Heloterä H, Tammela T, Benjamin LE, Ylä-Herttuala S, Leow CC, Koh GY, Alitalo K. Effects of Angiopoietin-2 blocking antibody on endothelial cell-cell junctions and lung metastasis. J Natl Cancer I, in press.

Heljasvaara R and Pihlajaniemi T. Experimental tumour models in mice. In: Mouse as a model organism – from animals to cells. Ed. Brakebusch C and Pihlajaniemi T. Springer-Verlag, 2011.

Brakebush C and Pihlajaniemi T. Mouse as a model organism – from animals to cells. Ed. Brakebusch C and Pihlajaniemi T. Springer-Verlag, 2011.

Pietilä R, Nätynki M, Tammela T, Kangas J, Pulkki KH, Limaye N, Vikkula M, Koh GY, Saharinen P, Alitalo K, Eklund L. Ligand oligomerization state controls Tie2 receptor trafficking and Angiopoietin-2 ligand-specific responses. J Cell Sci, in press.

Heikkinen A, Tu H, Pihlajaniemi T. Collagen XIII: a type II transmembrane protein with relevance to musculoskeletal tissues, microvessels and inflammation. Int J Biochem Cell B, in press.

Doctoral Theses 2011

Aino Kinnunen: Collagen XVIII regulates basement membrane integrity. Specific effects of its isoforms on the choroid plexus, kidney and hair follicle. Acta Universitatis Ouluensis D 1095, 2011.

Research Group Members

Project Leader:
Taina Pihlajaniemi, M.D., Ph.D., Professor, Vice President (Vice Rector) Provost for Science and Research (University of Oulu)

Senior and Post-doctoral Investigators:
Lauri Eklund, Ph.D. (Academy of Finland)
Harri Elamaa, Ph.D. (Academy of Finland)
Anne Heikkinen, Ph.D. (Academy of Finland)
Ritva Heljasvaara, Ph.D. (University of Oulu)
Pirkko Huhtala, Ph.D. (Biocenter Oulu and University of Oulu)
Merja Hurskainen, M.D., Ph.D. (University of Oulu) leave of absence
Valerio Izzi, Ph.D. (Academy of Finland)
Sanna-Maria Karppinen, Ph.D. (Academy of Finland)
Jarkko Koivunen, Ph.D. (Academy of Finland)
Karolina Rasi, M.D., Ph.D. (Academy of Finland)
Heli Ruotsalainen, Ph.D. (Academy of Finland)
Hongmin Tu, Ph.D. (Biocenter Oulu)

Ph.D. Students:
Miki Aho, Med.Cand. (Sigrid Jusélius Foundation)
Mari Aikio, M.Sc. (Biocenter Oulu Graduate School)
Ann-Marie Auvinen, Med.Cand. (Sigrid Jusélius Foundation and University of Oulu)
Raman Devarajan, M.Sc. (Academy of Finland)
Hanne-Kaisa Honkanen, Med.Cand. (University of Oulu and Sigrid Jusélius Foundation)
Heli Härönen, Med.Cand. . (Sigrid Jusélius Foundation and University of Oulu)
Jaakko Kangas, Med.Cand. (Academy of Finland and University of Oulu)
Inderjeet Kaur, Ph.D. (chemistry) (Sigrid Jusélius Foundation, Academy of Finland)
Antti Kemppainen, Med.Stud. (Sigrid Jusélius Foundation)
Vanessa López, M.Sc. (Biocenter Oulu)
Marjut Nätynki, M.Sc. (Academy of Finland)
Riikka Pietilä, M.Sc. (University of Oulu)
Mia Rinta-Jaskari, Med.Stud.  (Sigrid Jusélius Foundation)
Heidi Tanayama, Med.Cand. (Sigrid Jusélius Foundation and University of Oulu)
David Vicente, D.V.M. (Biocenter Oulu)
Zarin Zainul, M.Sc. (Academy of Finland)

Laboratory Technicians, 5 (University of Oulu, FIMM and Finnish Cancer Organizations)

Main source of salary in brackets.

Foreign Scientists, 8

National and International Activities

Visiting Researchers in 2011 (over two weeks)

Paul Janmey, Prof.: University of Pennsylvania, Philadelphia, USA
Lisette van Tassel, M.Sc.: University of Toronto, Mississauga, Ontario, Canada

EU Projects

Preparatory phase ESFRI project “The European infrastructure for phenotyping and archiving of model mammalian genomes (Infrafrontier)”. EU contract no. FP7-CP-CSA_INFRA-2007-2.2.01/grant agreement no. 211404.2008-2011. Partner.

COST action ”ECMNet: Brain extracellular matrix in health and disease”. Action no. BM1001 2010-2014. Management committee member Taina Pihlajaniemi.

NordForsk Projects

Researcher network project “NorIMM”. NordForsk contract no. 27840, 2010-2012. Coordinator Taina Pihlajaniemi.