Regulation of Epithelial Cell Functions by Integrins and the Extracellular Matrix

Project leader
Aki Manninen, Adjunct Professor of Cell Biology, Ph.D.
Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu

Background and Significance

The ECM, deposited by the cells themselves, not only scaffolds tissues but is also rich in regulatory signals that orchestrate cellular functions and responses. A laminin-rich basement membrane (BM) is of particular importance as it orients the polarity of epithelial cells by serving as a ligand for members of the integrin family of ECM receptors. Twenty-four different integrin heterodimers have been reported in humans. How the different integrins convey signals from the ECM into the cells is not thoroughly understood. However, it is clear that even subtle dysfunctions in this reciprocal communication can disrupt organ functionality. Therefore, it is important to build up comprehensive knowledge of the regulation of epithelial cell function by cell–ECM interactions.

Recent Progress and Future Goals

Dissecting the functional roles of specific integrins in epithelial cells
The bi-directional function of integrins enables ECM binding by the cell while simultaneously sensing its chemical composition and physical properties. Sites of integrin adhesion serve as localized organizing centres for the cytoskeleton, regulating changes in the cell architecture. Integrin adhesion sites are also sensitive to mechanical stimuli and capable of imparting contractility across the cytoskeleton, thereby facilitating processes such as cell spreading and migration. A clear link exists between integrin functions, cell polarity and epithelial morphogenesis. Moreover, integrins are central to development as well as to epithelial pathologies, but their specific roles at a cellular level as well as molecular machineries underlying their specific functions have not been thoroughly characterized. It is evident that even integrins with similar ligands have differing cellular functions governed via mechanisms beyond their ligand-binding specificity. A complex network of accompanying effector molecules at the cell membrane fine-tunes the cellular response. By utilizing comprehensive analysis of integrin associated complexes (IACs) we investigate the protein-protein interaction networks that exert the functions of specific integrins. The functional roles of selected integrin interacting candidate proteins are then studied in advanced in vitro cell/tissue culture models using a wide variety of cell biological and biochemical methods.

Integrin-mediated signals in the regulation of epithelial cell shape and morphogenesis
In three-dimensional (3D) culture environments MDCK cells recapitulate epithelial morphogenesis by forming polarized lumen-enclosing spherical cysts. By analysing the behaviour and phenotypes of integrin-knockdown (Itg-KD) cells in various culture conditions we have observed that α2β1- and β4-integrins are required for ECM recognition and provide cues for the establishment of laminin-based basal cues in collagen-I gels (Myllymäki et al., PLoSONE, 2011). α3β1-integrins were found to be essential for control of the cell division axis in the maintenance of lumen integrity in laminin-rich basement membrane extract (BME) gels (Myllymäki et al., PLoSONE, 2011). In our ongoing studies we are addressing the molecular mechanisms by which integrins transmit BM-derived signals to regulate epithelial cell polarization and morphogenesis. To better understand the molecular mechanisms underlining these phenomena, we isolate IACs from different integrin-mutant cell lines and employ a mass spectrometry-based approaches to identify critical IAC-components that mediate specific functions of BM-binding integrins.

αV-integrins regulate cellular mechanotransduction responses in epithelial cells
As mentioned above, integrins are crucial sensors and effectors of cellular biomechanics. Our RNAi-based screening approach revealed a central role for αV-integrins as regulators of the maturation of focal adhesions (FAs) in MDCK cells (Teräväinen et al., PLoSONE, 2013). When αV-integrins were depleted, MDCK cells became unable to correctly respond to changes in ECM stiffness. We are currently focusing on dissecting the molecular machinery associated with αV-integrins at maturing focal adhesions. Integrins lack enzymatic activity, so they rely on associated proteins which modulate cellular signal transduction pathways and thereby cellular functions. By using mainly proteomics-based approaches we aim to identify novel molecular effectors that are functionally associated with αV-integrins.

Specific integrin functions in prostate cancer
In several tumour types, the regulation of integrin-mediated signalling is perturbed, thereby contributing to abnormal migration, invasion, proliferation and survival of tumour cells. Although integrins themselves are not thought to transform cells, recent data demonstrate that several integrins contribute to tumorigenesis by inhibiting or cooperating with oncogenes. The vast majority of diagnosed cancers are carcinomas that originate from epithelium of skin, breast, prostate, colon or kidneys. Epithelial polarity and/or polarized organization of epithelial cells within tissues have emerged as critical regulators of oncogenesis and multiple polarity-related genes appear to function as tumour suppressors. In this project we study functional synergies or antagonisms between selected integrins (implicated in the regulation of epithelial polarity in our previous studies) with prostate cancer associated oncogenes and loss of tumour suppressors. 3D cultures of genetically engineered prostate epithelial cell lines as well as primary prostate organoids are used as a model system. Lentiviral vectors are used to overexpress (oncogenes) or knock out (CRISPR) selected prostate cancer associated genes. Our aim is to facilitate the development of novel targeted therapeutic approaches to inhibit malignant transformation by modulating reciprocal signalling between prostate cancer cells and their tumour-promoting microenvironment.

Selected Publications

Pitkäaho T, Manninen A, Naughton TJ (2019) Focus prediction in digital holographic microscopy using deep convolutional neural networks. Appl Opt 58 (5), A202-A208

Kozlova N, Mennerich D, Samoylenko A, Dimova EY, Koivunen P, Biterova E, Richter K, Hassinen A, Kellokumpu S, Manninen A, Miinalainen I, Glumoff V, Ruddock L, Drobot L, Kietzmann T (2018) The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2. bioRxiv doi:

Myllymäki SM, Kämäräinen UR, Liu X, Cruz SP, Miettinen S, Vuorela M, Varjosalo, M, Manninen A (2018). Assembly of the β4-integrin interactome based on proximal biotinylation in the presence and absence of heterodimerization. MCP Nov 7, doi: 10.1074/mcp.RA118.001095. [Epub ahead of print]

Nair RR, Koivisto H, Jokivarsi K, Miinalainen IJ, Autio KJ, Manninen A, Tanila H, Hiltunen JK, Kastaniotis AJ (2018) Impaired mitochondrial fatty acid synthesis leads to neurodegeneration in mice. J Neurosci. 38(45):9781-9800.
Paakkola T, Salokas K, Miinalainen I, Lehtonen S, Manninen A, Kaakinen M, Ruddock LW, Varjosalo M, Kaarteenaho R, Uusimaa J, Hinttala R. (2018) Biallelic mutations in human NHLRC2 enhance myofibroblast differentiation in FINCA disease. Hum Mol Genet. Sep 19, published ahead of print.

Gao P, Xia JH, Sipeky C, Dong XM, Zhang Q, Yang Y, Zhang P, Cruz SP, Zhang K, Zhu J, Lee HM, Suleman S, Giannareas N, Liu S, The PRACTICAL consortium, Tammela TLJ, Auvinen A, Wang X, Huang Q, Wang L, Manninen A, Vaarala MH, Wang L, Schleutker J, Wei GH (2018) Biology and clinical implications of the 19q13 aggressive prostate cancer susceptibility locus. Cell, 174:576-589.

Raykhel I, Moafi F, Myllymäki SM, Greciano PG, Matlin KS, Moyano JV, Manninen A*, Myllyharju J* (2018) Bambi is a novel HIF1-dependent modulator of TGFβ-mediated disruption of cell polarity in hypoxia. J Cell Sci, in press  doi: 10.1242/jcs.210906, Published 23 April 2018, *equal contribution

Uusimaa J, Kaarteenaho R, Paakkola T, Tuominen H, Karjalainen MK, Nadaf J, Varilo T, Uusi- Mäkelä M, Suo-Palosaari M, Ilkka Pietilä I, Hiltunen AE, Ruddock L, Alanen H, Biterova E, Miinalainen I, Salminen A, Soininen R, Manninen A, Sormunen R, Kaakinen M, Vuolteenaho R, Herva R, Vieira P,Dunder T, Kokkonen H,Moilanen JS, Rantala H, Nogee LM, Majewski J, Rämet M, Hallman M, Hinttala R (2018) NHLRC2 variants identified in patients with Fibrosis, Neurodegeneration, and Leptomeningeal Angiomatosis (FINLA) - Characterization of a novel  cerebropulmonary disease. Acta Neuropathologica, 135: 727-742.

Zhang K, Myllymäki SM, Gao P, Devarajan R, Kytölä V, Nykter M, Heljäsvaara R, Wei GH, Manninen A (2017) Oncogenic K-Ras upregulates ITGA6-expression via FOSL1 to induce anoikis resistance and synergizes with αV-class integrins to promote EMT. Oncogene, 36: 5681-5694.

Matlin KS, Myllymäki SM, Manninen A. (2017) Laminins in Epithelial Cell Polarization - Old Questions in Search of New Answers. Cold Spring Harbor Perspectives in Biology, 9: a027920.

Moilanen J, Löffek S, Kokkonen N, Salo S, Väyrynen J, Hurskainen T, Manninen A, Riihilä P, Heljasvaara R, Franzke CW, Kähäri VM, Salo T, Mäkinen M, Tasanen K (2017). Significant role of collagen XVII and integrin β4 in migration and invasion of the less aggressive squamous cell carcinoma cells. Scientific Reports, 7:45057.

Manninen A, Varjosalo M (2017) A proteomics view on integrin-mediated adhesions. Proteomics, 17: p.201600022.

Zhang K, Lee HM, Wei GH, Manninen A . Meta-analysis of gene expression and integrin-associated signaling pathways in papillary renal cell carcinoma subtypes. Oncotarget, 7: 84178-84189, 2016.

Carrera M, Bitu CC, de Oliveira CE, Cervigne NK, Graner E, Manninen A, Salo T, Coletta RD. HOXA10 controls proliferation, migration and invasion in oral squamous cell carcinoma. Int J Clin Exp Pathol 8(4):3613-23, 2015.

Cattavarayane S, Palovuori R, Jayendrakishore TR, Manninen A. α6β1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF. BMC Cell Biol 16:3, 2015.

Junttila S, Saarela U, Halt K, Manninen A, Pärssinen H, Lecca RM, Brandli AW, Sims-Lucas S, Skovorodkin I, Vainio SJ. Functional genetic targeting of the embryonic kidney progenitor cells ex vivo. J Am Soc Nephrol 26(5):1126-37, 2015.

Manninen A. Epithelial polarity - Generating and integrating signals from the ECM with integrins. Exp Cell Res 334(2): 337-349, 2015.

Teräväinen TP, Myllymäki SM, Friedrichs J, Strohmeyer N, Moyano JV, Wu C, Matlin KS, Muller DJ, Manninen A. αV-integrins are required for mechanotransduction in MDCK epithelial cells. PLoS One. 2013. 8(8):e71485.

Huang Q, Whitington T, Gao P, Lindberg JF,  Yuehong Yang Y,  Sun J, Väisänen MR, Szulkin R,  Annala M, Yan J, Egevad LA, Zhang K, Lin R, Jolma A, Nykter M, Manninen A, Wiklund F, Vaarala MH, Visakorpi T, Xu J, Taipale J and Wei GH. A prostate cancer susceptibility allele at 6q22 increases RFX6 expression by modulating HOXB13 chromatin binding. Nature Genetics. 2014. 46,126–135.

Veikkolainen V, Naillat F, Railo A, Chi L, Manninen A, Hohenstein P, Hastie N, Vainio S, Elenius K. ErbB4 modulates tubular cell polarity and lumen diameter during kidney development. J Am Soc Nephrol 23:112-122, 2012.

Greciano PG, Moyano JV, Buschmann MM, Tang J, Lu Y, Rudnicki J, Manninen A, Matlin KS. Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells. Mol Biol Cell 23:121-136, 2012.

Rilla K, Pasonen-Seppänen S, Kärnä R, Karjalainen HM, Törrönen K, Koistinen V, Tammi MI, Tammi RH, Teräväinen T, Manninen A. HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium. Histochem Cell Biol 137:153-164, 2012.

de Oliveira JT, de Matos AJ, Santos AL, Pinto R, Gomes J, Hespanhol V, Chammas R, Manninen A, Bernardes ES, Reis CA, Rutteman G, Gärtner F. Sialylation regulates galectin-3/ligand interplay during mammary tumour progression - a case of targeted uncloaking. Int J Dev Biol 55:823-834, 2011.

Moilanen A-M, Rysä J, Mustonen E, Serpi R, Aro J, Tokola H, Leskinen H, Manninen A, Levijoki J, Vuolteenaho O, Ruskoaho H. Intramyocardial BNP gene delivery improves cardiac function through distinct context-dependent mechanisms. Circ Heart Fail 4:483-495, 2011.

Myllymäki SM, Teräväinen TP, Manninen A. Two distinct integrin-mediated mechanisms contribute to apical lumen formation in epithelial cells. PLoS One 6:e19453, 2011.

de Oliveira,J.T., de Matos,A.J., Gomes,J., Vilanova,M., Hespanhol,V., Manninen,A., Rutteman,G., Chammas,R., de Fatima Gartner,M., Soares Bernardes,E. Coordinated expression of galectin-3 and galectin-3-binding sites in malignant mammary tumours: implications for tumour metastasis. Glycobiology, 20(11):1341-52, 2010.

Friedrichs J, Manninen A, Muller DJ, Helenius J. Galectin-3 regulates integrin a2b1-mediated adhesion to collagen-I and ­IV. J Biol Chem, 283(47):32264-72, 2008.

Torkko JM, Manninen A, Schuck S, Simons K. Depletion of apical transport proteins perturbs epithelial cyst formation and ciliogenesis. J Cell Sci, 121(8):1193-203, 2008.

Friedrichs J, Torkko JM, Helenius J, Teravainen TP, Fullekrug J, Muller DJ, Simons K, Manninen A. Contributions of galectin-3 and -9 to epithelial cell adhesion analysed by single cell force spectroscopy. J Biol Chem, 282( 40): 29375-83, 2007.

Schuck S, Gerl MJ, Ang A, Manninen A, Keller P, Mellman I, Simons K. Rab10 is involved in basolateral transport in polarized Madin-Darby canine kidney cells. Traffic 8:47-60, 2007.

Manninen A, Verkade P, Le Lay S, Torkko J, Kasper M, Fullekrug J, Simons K. Caveolin-1 is not essential for biosynthetic apical membrane transport. Mol Cell Biol 25:10087-10096, 2005.

Vieira O, Verkade P, Manninen A, Simons K. FAPP2 is involved in the transport of apical cargo in polarized MDCK cells. J Cell Biol 170:521-526, 2005.

Delacour D, Gouyer V, Zanetta J-P, Drobecq H, Leteurtre E, Grard G, Moreau-Hannedouche O, Maes E, Pons A, Andre S, Le Bivic A, Gabius HJ, Manninen A, Simons K, Huet G. Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells. J Cell Biol 169:491-501, 2005.

Schuck S, Manninen A, Honsho M, Fullekrug J, Simons K. Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference. Proc Natl Acad Sci USA 101:4912-4917, 2004.

Doctoral Theses

Satu-Marja Myllymäki: Specific roles of epithelial integrins in chemical and physica I sensing of the extracellular matrix to regulate cell shape and polarity. Acta Universitatis Ouluensis. D, Medica 1308.

Sandhananakrishnan Cattavarayane: Regulation of murine embryonic stem cell self-renewal by integrin-extracellular matrix interactions. Faculty of Biochemistry and Molecular Medicine Thesis Series 4069.

Kai Zhang: Specific integrin functions regulating the interplay between cancer cells and their microenvironment.

Research Group Members

Project Leader:
Aki Manninen, Ph.D. (Academy of Finland, Biocenter Oulu, UO strategic funding targeted for BF operations and University of Oulu)

Senior and Post-doctoral Investigators:
Irina Raykhel, Ph.D. (Biocenter Oulu, UO strategic funding targeted for BF operations and University of Oulu)

Ph.D. students:
Sara Cruz, M.Sc. (University of Oulu)

Laboratory Technicians:
Jaana Träskelin, part-time (Biocenter Oulu)
Riitta Jokela (University of Oulu)

Main source of salary in brackets.

Foreign Scientists, 2

Last updated: 21.1.2019