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Fibulin-2 is involved in early extracellular matrix development of the outgrowing mouse mammary epithelium

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Abstract

Cell–matrix interactions control outgrowth of mammary epithelium during puberty and pregnancy. We demonstrate here that the glycoprotein fibulin-2 (FBLN2) is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth. FBLN2 was specifically localized to the cap cells of the terminal end buds during puberty and to myoepithelial cells during very early pregnancy (days 2–3) even before morphological changes to the epithelium become microscopically visible, but was down-regulated thereafter. Exposure to exogenous oestrogen (E2) or E2 plus progesterone (P) increased Fbln2 mRNA expression in the pubertal gland, indicating hormonal control. FBLN2 was co-expressed and co-localised with the proteoglycan versican (VCAN) and co-localised with laminin (LN), while over-expression of FBLN2 in HC-11 cells increased cell adhesion to several extracellular matrix proteins including LN and fibronectin, but not collagens. Mammary glands from Fbln2 knockout mice showed no obvious phenotype but increased fibulin-1 (FBLN1) staining was detected, suggesting a compensatory mechanism by other fibulin family members. We hypothesise that similar to embryonic aortic smooth muscle development, FBLN2 and VCAN expression alters the cell–matrix interaction to allow mammary ductal outgrowth and development during puberty and to enable epithelial budding during pregnancy.

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Abbreviations

BM:

Basement membrane

CMV:

Cytomegalovirus

Ctrl:

Control

DAPI:

4′,6-diamidino-2-phenylindole

DMEM:

Dulbecco’s Modified Eagle’s Medium

ECM:

Extracellular matrix

E2:

Estradiol

EGF:

Epidermal growth factor

ER:

Oestrogen receptor

FBS:

Foetal bovine serum

FFPE:

Formalin-fixed paraffin-embedded

FN:

Fibronectin

Fwd:

Forward

HRP:

Horseradish peroxidase

IF:

Immunofluorescence

IgG:

Immunoglobulin G

IHC:

Immunohistochemistry

KO:

Knock-out

Krt:

Cytokeratin

LN111:

Laminin-111

LN332:

Laminin-332

LTBP1:

Latent TGFβ binding protein 1

P:

Progesterone

Post-LN:

Post-lymph node (area beyond major mammary lymph node)

Pre-LN:

Pre-lymph node (area between nipple and major mammary lymph node)

qRT-PCR:

Quantitative reverse transcriptase polymerase chain reaction

rev:

Reverse

RGD:

Argine, glycine, aspartate

RIPA:

Radio immunoprecipitation assay

RPMI:

Roswell Park Memorial Institute

SD:

Standard deviation

SEM:

Standard error of the mean

TEB:

Terminal end bud

TGFβ:

Transforming growth factor beta

VCAN:

Versican

References

  1. Muschler J, Streuli CH (2010) Cell-matrix interactions in mammary gland development and breast cancer. Cold Spring Harb Perspect Biol 2:a003202

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Daniel CW, Silberstein GB, Strickland P (1987) Direct action of 17 beta-estradiol on mouse mammary ducts analyzed by sustained release implants and steroid autoradiography. Cancer Res 47:6052–6057

    CAS  PubMed  Google Scholar 

  3. Green KA, Lund LR (2005) ECM degrading proteases and tissue remodelling in the mammary gland. BioEssays 27:894–903

    Article  CAS  PubMed  Google Scholar 

  4. Argraves WS, Dickerson K, Burgess WH, Ruoslahti E (1989) Fibulin, a novel protein that interacts with the fibronectin receptor beta subunit cytoplasmic domain. Cell 58:623–629

    Article  CAS  PubMed  Google Scholar 

  5. Roark EF, Keene DR, Haudenschild CC, Godyna S, Little CD et al (1995) The association of human fibulin-1 with elastic fibers: an immunohistological, ultrastructural, and RNA study. J Histochem Cytochem 43:401–411

    Article  CAS  PubMed  Google Scholar 

  6. Miosge N, Gotz W, Sasaki T, Chu ML, Timpl R et al (1996) The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo. Histochem J 28:109–116

    Article  CAS  PubMed  Google Scholar 

  7. de Vega S, Iwamoto T, Yamada Y (2009) Fibulins: multiple roles in matrix structures and tissue functions. Cell Mol Life Sci 66:1890–1902

    Article  PubMed  Google Scholar 

  8. Kobayashi N, Kostka G, Garbe JH, Keene DR, Bachinger HP et al (2007) A comparative analysis of the fibulin protein family. Biochemical characterization, binding interactions, and tissue localization. J Biol Chem 282:11805–11816

    Article  CAS  PubMed  Google Scholar 

  9. Sasaki T, Gohring W, Pan TC, Chu ML, Timpl R (1995) Binding of mouse and human fibulin-2 to extracellular matrix ligands. J Mol Biol 254:892–899

    Article  CAS  PubMed  Google Scholar 

  10. Sasaki T, Wiedemann H, Matzner M, Chu ML, Timpl R (1996) Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix. J Cell Sci 109(Pt 12):2895–2904

    CAS  PubMed  Google Scholar 

  11. Reinhardt DP, Sasaki T, Dzamba BJ, Keene DR, Chu ML et al (1996) Fibrillin-1 and fibulin-2 interact and are colocalized in some tissues. J Biol Chem 271:19489–19496

    Article  CAS  PubMed  Google Scholar 

  12. Loveland K, Schlatt S, Sasaki T, Chu ML, Timpl R et al (1998) Developmental changes in the basement membrane of the normal and hypothyroid postnatal rat testis: segmental localization of fibulin-2 and fibronectin. Biol Reprod 58:1123–1130

    Article  CAS  PubMed  Google Scholar 

  13. Zhang RZ, Pan TC, Zhang ZY, Mattei MG, Timpl R et al (1994) Fibulin-2 (FBLN2): human cDNA sequence, mRNA expression, and mapping of the gene on human and mouse chromosomes. Genomics 22:425–430

    Article  CAS  PubMed  Google Scholar 

  14. Ng KM, Catalano MG, Pinos T, Selva DM, Avvakumov GV et al (2006) Evidence that fibulin family members contribute to the steroid-dependent extravascular sequestration of sex hormone-binding globulin. J Biol Chem 281:15853–15861

    Article  CAS  PubMed  Google Scholar 

  15. Fassler R, Sasaki T, Timpl R, Chu ML, Werner S (1996) Differential regulation of fibulin, tenascin-C, and nidogen expression during wound healing of normal and glucocorticoid-treated mice. Exp Cell Res 222:111–116

    Article  CAS  PubMed  Google Scholar 

  16. Ström A, Olin AI, Aspberg A, Hultgardh-Nilsson A (2006) Fibulin-2 is present in murine vascular lesions and is important for smooth muscle cell migration. Cardiovasc Res 69:755–763

    Article  PubMed  Google Scholar 

  17. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407:313–319

    Article  CAS  PubMed  Google Scholar 

  18. Sicot FX, Tsuda T, Markova D, Klement JF, Arita M et al (2008) Fibulin-2 is dispensable for mouse development and elastic fiber formation. Mol Cell Biol 28:1061–1067

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Morris JS, Stein T, Pringle MA, Davies CR, Weber-Hall S et al (2006) Involvement of axonal guidance proteins and their signaling partners in the developing mouse mammary gland. J Cell Physiol 206:16–24

    Article  CAS  PubMed  Google Scholar 

  20. Stein T, Morris JS, Davies CR, Weber-Hall SJ, Duffy M-A et al (2004) Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3. Breast Cancer Res 6:R75–R91

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Pan TC, Sasaki T, Zhang RZ, Fassler R, Timpl R et al (1993) Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding. J Cell Biol 123:1269–1277

    Article  CAS  PubMed  Google Scholar 

  22. Sasaki T, Kostka G, Gohring W, Wiedemann H, Mann K et al (1995) Structural characterization of two variants of fibulin-1 that differ in nidogen affinity. J Mol Biol 245:241–250

    Article  CAS  PubMed  Google Scholar 

  23. Detre S, Saclani Jotti G, Dowsett M (1995) A “quickscore” method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. J Clin Pathol 48:876–878

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Okada H, Nakajima T, Yoshimura T, Yasuda K, Kanzaki H (2003) Microarray analysis of genes controlled by progesterone in human endometrial stromal cells in vitro. Gynecol Endocrinol 17:271–280

    Article  CAS  PubMed  Google Scholar 

  25. Gallagher WM, Currid CA, Whelan LC (2005) Fibulins and cancer: friend or foe? Trends Mol Med 11:336–340

    Article  CAS  PubMed  Google Scholar 

  26. Miosge N, Sasaki T, Chu ML, Herken R, Timpl R (1998) Ultrastructural localization of microfibrillar fibulin-1 and fibulin-2 during heart development indicates a switch in molecular associations. Cell Mol Life Sci 54:606–613

    Article  CAS  PubMed  Google Scholar 

  27. Van Keymeulen A, Rocha AS, Ousset M, Beck B, Bouvencourt G et al (2011) Distinct stem cells contribute to mammary gland development and maintenance. Nature 479:189–193

    Article  PubMed  Google Scholar 

  28. Silberstein GB, Daniel CW (1982) Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct. Dev Biol 90:215–222

    Article  CAS  PubMed  Google Scholar 

  29. Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AH et al (2000) Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to L-selectin, P-selectin, and CD44. J Biol Chem 275:35448–35456

    Article  CAS  PubMed  Google Scholar 

  30. Bajorath J, Greenfield B, Munro SB, Day AJ, Aruffo A (1998) Identification of CD44 residues important for hyaluronan binding and delineation of the binding site. J Biol Chem 273:338–343

    Article  CAS  PubMed  Google Scholar 

  31. Evanko SP, Angello JC, Wight TN (1999) Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 19:1004–1013

    Article  CAS  PubMed  Google Scholar 

  32. Olin AI, Morgelin M, Sasaki T, Timpl R, Heinegard D et al (2001) The proteoglycans aggrecan and Versican form networks with fibulin-2 through their lectin domain binding. J Biol Chem 276:1253–1261

    Article  CAS  PubMed  Google Scholar 

  33. Zhang HY, Chu ML, Pan TC, Sasaki T, Timpl R et al (1995) Extracellular matrix protein fibulin-2 is expressed in the embryonic endocardial cushion tissue and is a prominent component of valves in adult heart. Dev Biol 167:18–26

    Article  CAS  PubMed  Google Scholar 

  34. Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR (1998) The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Dev Biol 202:56–66

    Article  CAS  PubMed  Google Scholar 

  35. Radisky ES, Radisky DC (2010) Matrix metalloproteinase-induced epithelial-mesenchymal transition in breast cancer. J Mammary Gland Biol Neoplasia 15:201–212

    Article  PubMed Central  PubMed  Google Scholar 

  36. Isogai Z, Aspberg A, Keene DR, Ono RN, Reinhardt DP et al (2002) Versican interacts with fibrillin-1 and links extracellular microfibrils to other connective tissue networks. J Biol Chem 277:4565–4572

    Article  CAS  PubMed  Google Scholar 

  37. Ohno-Jinno A, Isogai Z, Yoneda M, Kasai K, Miyaishi O et al (2008) Versican and fibrillin-1 form a major hyaluronan-binding complex in the ciliary body. Invest Ophthalmol Vis Sci 49:2870–2877

    Article  PubMed  Google Scholar 

  38. Williams JM, Daniel CW (1983) Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev Biol 97:274–290

    Article  CAS  PubMed  Google Scholar 

  39. Tsuda T, Wang H, Timpl R, Chu ML (2001) Fibulin-2 expression marks transformed mesenchymal cells in developing cardiac valves, aortic arch vessels, and coronary vessels. Dev Dyn 222:89–100

    Article  CAS  PubMed  Google Scholar 

  40. Meier-Abt F, Milani E, Roloff T, Brinkhaus H, Duss S et al (2013) Parity induces differentiation and reduces Wnt/Notch signaling ratio and proliferation potential of basal stem/progenitor cells isolated from mouse mammary epithelium. Breast Cancer Res 15:R36

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Chapman SL, Sicot FX, Davis EC, Huang J, Sasaki T et al (2010) Fibulin-2 and fibulin-5 cooperatively function to form the internal elastic lamina and protect from vascular injury. Arterioscler Thromb Vasc Biol 30:68–74

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Kostka G, Giltay R, Bloch W, Addicks K, Timpl R et al (2001) Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice. Mol Cell Biol 21:7025–7034

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. Pfaff M, Sasaki T, Tangemann K, Chu ML, Timpl R (1995) Integrin-binding and cell-adhesion studies of fibulins reveal a particular affinity for alpha IIb beta 3. Exp Cell Res 219:87–92

    Article  CAS  PubMed  Google Scholar 

  44. Maller O, Martinson H, Schedin P (2010) Extracellular matrix composition reveals complex and dynamic stromal-epithelial interactions in the mammary gland. J Mammary Gland Biol Neoplasia 15:301–318

    Article  PubMed  Google Scholar 

  45. Utani A, Nomizu M, Yamada Y (1997) Fibulin-2 binds to the short arms of laminin-5 and laminin-1 via conserved amino acid sequences. J Biol Chem 272:2814–2820

    Article  CAS  PubMed  Google Scholar 

  46. Klinowska TC, Soriano JV, Edwards GM, Oliver JM, Valentijn AJ et al (1999) Laminin and beta1 integrins are crucial for normal mammary gland development in the mouse. Dev Biol 215:13–32

    Article  CAS  PubMed  Google Scholar 

  47. Longmate WM, Monichan R, Chu ML, Tsuda T, Mahoney MG, et al. (2014) Reduced Fibulin-2 contributes to loss of basement membrane integrity and skin blistering in mice lacking integrin alpha3beta1 in the epidermis. J Invest Dermatol

  48. Ono RN, Sengle G, Charbonneau NL, Carlberg V, Bachinger HP et al (2009) Latent transforming growth factor beta-binding proteins and fibulins compete for fibrillin-1 and exhibit exquisite specificities in binding sites. J Biol Chem 284:16872–16881

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Miyazono K, Olofsson A, Colosetti P, Heldin CH (1991) A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1. EMBO J 10:1091–1101

    CAS  PubMed Central  PubMed  Google Scholar 

  50. Zhang H, Wu J, Dong H, Khan SA, Chu ML et al (2014) Fibulin-2 deficiency attenuates angiotensin II-induced cardiac hypertrophy by reducing transforming growth factor-beta signalling. Clin Sci (Lond) 126:275–288

    Article  CAS  Google Scholar 

  51. Kouros-Mehr H, Werb Z (2006) Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis. Dev Dyn 235:3404–3412

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

The authors would like to thank Drs. Meier-Abt and Momo Bentires-Alj for sharing unpublished data, Dr JM Iglesias for sharing his technical expertise, and Jennifer Joyce for her technical assistance and providing the whole-mounts of the Fbln2 KO mice. All animal work was carried out under personal licence PIL 60/11179 and project licence PPL 60/3712 and was approved by the University of Glasgow ethics committee. The work was funded by a Breakthrough Breast Cancer project grant to BAG, who designed the initial study, and by the Egyptian Ministry for Higher Education to AMI and TS.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Institute of Cancer Sciences, College of MVLS, University of Glasgow, Glasgow, G12 8QQ, UK

    D. Olijnyk, A. M. Ibrahim, B. A. Gusterson & T. Stein

  2. Zoology Department, Faculty of Science, Cairo University, Giza, 12613, Egypt

    A. M. Ibrahim

  3. MVLS Pathology Unit Pathology Department, Southern General Hospital, Glasgow, G51 4TF, UK

    R. K. Ferrier

  4. Nemours Biomedical Research and Nemours Cardiac Center, Alfred I. duPont Hospital for Children, Wilmington, 19803, USA

    T. Tsuda

  5. Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA

    M.-L. Chu

  6. School of Veterinary Medicine, College of MVLS, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK

    J. S. Morris

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  1. D. Olijnyk
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  2. A. M. Ibrahim
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  3. R. K. Ferrier
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  4. T. Tsuda
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  5. M.-L. Chu
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  7. T. Stein
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Corresponding authors

Correspondence to T. Stein or J. S. Morris.

Additional information

T. Stein and J.S. Morris contributed equally to this project.

Electronic supplementary material

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18_2014_1577_MOESM1_ESM.pdf

Supplementary Figure 1: Fbln2 mRNA expression over development normalised to Krt14. Mean expression of Fbln2 (relative to puberty time point) in whole glands collected at puberty, adulthood, pregnancy, lactation and involution measured by qRT-PCR and normalised to Krt14. Error bars represent standard deviation (S.D.) between three replicate PCR reactions (PDF 102 kb)

18_2014_1577_MOESM2_ESM.pdf

Supplementary Figure 2: 3D reconstruction of pubertal TEB and ducts stained with anti-FBLN2 antibody. 3D reconstruction of the series of confocal microscopy images of transverse sections through the TEB and longitudinal sections through the ducts of a 6 week mouse mammary gland. 3D reconstruction was performed using Velocity software; images acquired every 0.7-0.8 μm. FBLN2 staining in the TEB was restricted to cap cells and the BM region (white arrow). Duct epithelium was negative for FBLN2 expression. Scale bars are 20 μm (PDF 64 kb)

18_2014_1577_MOESM3_ESM.pdf

Supplementary Figure 3: FBLN2, VCAN and LN111 co-localise in cap cells during puberty. (a) Low (left) and high (right) power images of FBLN2, VCAN and LN111 staining in consecutive formalin-fixed paraffin-embedded (FFPE)-sections of a pubertal mammary gland. FBLN2 and VCAN showed a nearly identical staining pattern, and all three proteins showed preferential expression in and around TEB cap cells, but very low expression in subtending ducts. Images are representative of results from three independent glands. (b) Close-up of FBLN2, VCAN, and LN111 staining in the cap cells and surrounding stroma of a TEB. Scale bars are 100 μm (PDF 491 kb)

18_2014_1577_MOESM4_ESM.pdf

Supplementary Figure 4: FBLN2 and VCAN co-localise to mammary ductal bud during early pregnancy. High power images of FBLN2 and VCAN immunohistochemical staining from a mammary gland at 3-days pregnancy. Both FBLN2 and VCAN localise preferentially to the myoepithelium of what appears to be a ductal bud. Scale bar 100 μm (PDF 141 kb)

18_2014_1577_MOESM5_ESM.pdf

Supplementary Figure 5: Potential binding partners for FBLN2 in the pubertal mouse mammary gland. Selection of FBLN2 binding partners and their cellular compartmentalization as described in the literature. All 14 proteins interact with FBLN2 in different experimental systems. Pubertal gene expression was examined using Post-LN up-regulated gene set (Post-LN vs Pre-LN) and TEB up-regulated gene set (TEB vs ducts). Genes identified as up-regulated in Post-LN strips and TEB are marked as yellow and blue circles respectively. White circles denote genes showing unaltered expression in the Post-LN and Pre-LN strips (PDF 41 kb)

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Olijnyk, D., Ibrahim, A.M., Ferrier, R.K. et al. Fibulin-2 is involved in early extracellular matrix development of the outgrowing mouse mammary epithelium. Cell. Mol. Life Sci. 71, 3811–3828 (2014). https://doi.org/10.1007/s00018-014-1577-4

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