Donald Gullberg Lab
PROJECTS IN THE GULLBERG LABORATORY
The Gullberg laboratory has characterized the integrin α11β1, which is expressed on subsets of normal fibroblasts and on carcinoma-associated fibroblasts. Cells lacking α11β1 display disturbed cell-collagen interactions, altered metalloproteinase synthesis and reduced cell proliferation. Major projects within the group aim to further understand the role of this collagen receptor and other fibroblast integrins during health and in disease.
Cells are anchored in the extracellular matrix (ECM) via specific receptors belonging to different superfamilies, including the integrin family. Out of the 24 integrin heterodimers, α1β1, α2β1, α10β1 and α11β1 integrins act as primary receptors for native collagens 1. ECM, integrins and the fibroblast are increasingly being recognized as being important in the control of the microenvironment 2. The ECM, in addition to its supportive structural role, acts as a reservoir for growth factors, guides cell migration, influences cell signaling, cell growth, cell differentiation and direct angiogenesis. In normal epithelial tissue, the stromal compartment provides the important support for the normal function of the epithelium and other cells constituting an organ. The normal stroma is composed of orderly structured mesenchymal cells (including fibroblasts) and extracellular substances, vascular and lymphatic networks, and minimal immune cell infiltrate. Fibroblasts are cells of mesodermal or ectomesenchymal origin that reside in every tissue of the body. Sampling of fibroblasts from different location in the body has revealed that fibroblasts are characterized by a positional code 3. In addition, rather than being cells of a defined fixed phenotype, they appear to be heterogeneous, even within tissues like skin 4. Under certain conditions fibroblasts can be activated and differentiate into so-called myofibroblasts, which are contractile collagen-producing cells 5. This differentiation occurs during wound healing, fibrosis, and the desmoplastic reaction in the tumor stroma.
The traditional view of the fibroblast as that of a rather passive cell type that merely produces the constituents of the interstitial extracellular matrix, is now changing. This is in part the result from work in the field of tumor biology where a paradigm shift has occurred so that there is now a widespread understanding of the importance of the tumor microenvironment for tumorigenesis and tumor metastasis. One of the major cell types that seem to be important for conditioning the microenvironment is the fibroblast. The emerging view suggests that fibroblasts play a much more active part in maintaining tissue homeostasis and sustaining certain dynamic tissue events, than previously thought. In tumors, the stroma is an activated tissue, i.e. mechanisms are initiated that support dynamic and active tissue remodeling 2. The activated status of many cells in the tumor stroma persists and has been likened to that of a non-healing wound 6. The changes that occur in the stroma during carcinogenesis include induction of fibroblasts proliferation, differentiation into myofibroblasts 5, altered amount and arrangement of stromal collagen, angiogenesis and increased immune and inflammatory cell infiltrates. Such changes are also reflected at biochemical and gene expression levels, as revealed by microarray studies comparing organ specific cancer versus normal tissues. Recent studies indicate that such changes in the stroma are not merely a bystander phenomenon, but play major roles in the process of carcinogenesis 7, including tumor cell growth, invasion, metastases, angiogenesis, and chemoresistance 8-10. The detailed mechanisms of these processes, which are likely to be tumor and tissue specific, are however, still largely unknown.
In an effort to develop novel cancer therapeutics, there is currently intensive effort to better understand the dynamic interplay between the tumor stroma, ECM, fibroblasts, endothelial cells and immune response cells. Given the central role of the activated carcinoma-associated fibroblasts (CAFs) in tumor growth, it is of interest to target the tumor fibroblast in anti-fibrogenesis approaches. This is the focus of a FUGE II-supported project in our laboratory. In this work we are establishing spheroids as a model system to study tumor-stroma interactions.
In our work with collagen-binding integrins, we identified a novel integrin subunit, named integrin α11, which has been the focus of our work for more than 10 years 11-14. Mice lacking this integrin are dwarfed, due to defective tooth structure, in turn reflecting a need for α11β1 on periodontal ligament fibroblasts during tooth eruption 15. In our continued work with this integrin it has become increasingly clear that α11β1 integrin might be a rather unique fibroblast marker From work with our existing mouse models, data is accumulating to suggest that α11β1 is functionally important in regulating tumor growth 16. From the tumor model, existing data suggest that a11 can work in a paracrine mode regulating IGF-II secretion 16. Some exciting years are thus ahead of us when the further detailed molecular mechanisms of α11β1 action need to be clarified. In these studies, subjecting our existing knockout mouse model to different challenging protocols as well as producing new animal models, which can validate and complement existing knockout mouse models, will be essential.
3.1 Characterization of the integrin α11 promoter in vitro and in vivo
To experimentally determine the nature of the sequences directing cell-, tissue- and differentiation-specific transcription, 3 kb of the human integrin α11 promoter sequence has been isolated and used to generate luciferase constructs containing different parts of the promoter. For in vitro analyses we have partly analyzed the human ITGA11 promoter in HT1080 and in primary human fibroblasts. Analysis of the promoter reveals potential binding sites for a number of transcription factors and experimental data indicate the importance of tandem Sp1 sites and an Ets-1-like site in regulating the basal proximal promoter 17. We have also mapped a TGF-β response elopement and the characterization of the promoter continues.
3.2 Studies of α11 promoter in tumor stroma and α11 promoter-directed deletion of tumor stroma genes
In our work with the promoter we have realized that the α11 promoter might be a good tool for fibroblast-specific Cre-mediated gene inactivation. We will characterize ITGA11 promoter in the tumor stroma. We are making a transgenic mouse strain overexpressing Cre-recombinase driven by this part of the α11 promoter.
3. 3 Generation of new transgenic models to study integrin function in fibroblasts
To further characterize the role of collagen-binding integrins using mouse models we are taking two approaches:
- 3.3.1 Establishment of an orthotopic colon carcinoma model in α11-/- nude mice.
- 3.3.2 Generating transgenic founder mice overexpressing integrin α11 chain
3.4 Use if tumor spheroids to study tumor-stroma interactions
In a FUGE II supported project we study how lung A549 cells interact with fibroblasts of different integrin and protoeglycan repertoires. We have set up a system of mixing tumor cells and fibroblasts of different ratios and in effect create mini tumors.
3.5 Characterization of zebrafish integrin α11
We have isolated cDNA for a zebrafish orthologue of α11, expressed the protein in mammalian cells, and have used sequence comparisons to identify evolutionary conserved residues which are being used to study importance of individual amino acids in integrin α11.
We focus our own work and collaborative efforts on the integrin α11β1, which recently has been shown to be involved in fibrosis, wound healing and tumorigenesis. In the current research plan I describe how we want to continue and build the competence around the detailed molecular mechanism of action of this receptor. The generation of a mouse model that facilitates gene deletion only in α11-expressing fibroblasts will make it feasible to selectively delete genes in tumor stroma fibroblasts and this work is predicted to have great potential in the field of tumor-stroma interactions. In summary, we believe that our work will lead to a better understanding of the contribution of integrin-mediated events for tissue function during physiological and pathophysiological conditions.
1. Popova, S.N., Lundgren-Akerlund, E., Wiig, H. & Gullberg, D. Physiology and pathology of collagen receptors. Acta Physiol (Oxf) 190, 179-87 (2007).
2. Radisky, D., Muschler, J. & Bissell, M.J. Order and disorder: the role of extracellular matrix in epithelial cancer. Cancer Invest 20, 139-53 (2002).
3. Rinn, J.L., Bondre, C., Gladstone, H.B., Brown, P.O. & Chang, H.Y. Anatomic demarcation by positional variation in fibroblast gene expression programs. PLoS Genet 2, e119 (2006).
4. Sorrell, J.M., Baber, M.A. & Caplan, A.I. Clonal characterization of fibroblasts in the superficial layer of the adult human dermis. Cell Tissue Res 327, 499-510 (2007).
5. Hinz, B. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol 127, 526-37 (2007).
6. Dvorak, H.F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 315, 1650-9 (1986).
7. Bissell, D.M. Chronic liver injury, TGF-beta, and cancer. Exp Mol Med 33, 179-90 (2001).
8. Nakamura, N. et al. Phenotypic differences of proliferating fibroblasts in the stroma of lung adenocarcinoma and normal bronchus tissue. Cancer Sci 95, 226-32 (2004).
9. Muerkoster, S. et al. Tumor stroma interactions induce chemoresistance in pancreatic ductal carcinoma cells involving increased secretion and paracrine effects of nitric oxide and interleukin-1beta. Cancer Res 64, 1331-7 (2004).
10. Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. in Cell, Vol. 121 335-48 (2005).
11. Velling, T., Kusche-Gullberg, M., Sejersen, T. & Gullberg, D. cDNA cloning and chromosomal localization of human α11 integrin. A collagen-binding, I domain-containing, β1-associated integrin α-chain present in muscle tissues. J. Biol. Chem. 274, 25735-25742 (1999).
12. Tiger, C.F., Fougerousse, F., Grundstrom, G., Velling, T. & Gullberg, D. alpha11beta1 integrin is a receptor for interstitial collagens involved in cell migration and collagen reorganization on mesenchymal nonmuscle cells. Dev Biol 237, 116-29 (2001).
13. Popova, S.N. et al. The mesenchymal α11β1 integrin attenuates PDGF-BB-stimulated chemotaxis of embryonic fibroblasts on collagens. Dev. Biol. 270, 427-442 (2004).
14. Gullberg, D., Velling, T., Sjoberg, G. & Sejersen, T. Up-regulation of a novel integrin alpha-chain (αmt) on human fetal myotubes. Dev. Dyn. 204, 57-65 (1995).
15. Popova, S.N. et al. Alpha11 beta1 integrin-dependent regulation of periodontal ligament function in the erupting mouse incisor. Mol Cell Biol 27, 4306-16 (2007).
16. Zhu, C.Q. et al. Integrin alpha 11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells. Proc Natl Acad Sci U S A 104, 11754-9 (2007).
17. Lu, N., Heuchel, R., Barczyk, M., Zhang, W.M. & Gullberg, D. Tandem Sp1/Sp3 sites together with an Ets-1 site cooperate to mediate alpha11 integrin chain expression in mesenchymal cells. Matrix Biol 25, 118-29 (2006).