Daniel S. Greenspan
BA, New York University
PhD, New York University
Postdoctoral Research, Yale University
4503 WIMR II
1111 Highland Ave
Madison, WI 53705
Extracellular modulators of cellular behaviors in development, homeostasis, and disease
Our focus has been on genes and gene products important to
vertebrate development and human disease. We have previously discovered a number of
developmentally important genes, and determined their involvement in normal morphogenesis and
in genetic abnormalities. For, example, we were involved in isolating and characterizing the locus for the
disfiguring and ultimately fatal heritable skin disorder dystrophic epidermolysis bullosa.
We've also found and helped characterize the locus for classic Ehlers-Danlos syndrome, which occurs in about
1 in 20,000 births and produces individuals with fragile connective tissues.
Much work in the lab currently focuses on extracellular regulatory proteins that control and
orchestrate events as diverse as growth factor signaling and formation of the extracellular matrix (ECM). Such events are critical in
adult vertebrates (e.g. for maintenance of the cardiovascular system, wound healing, turnover of the skeleton and synaptic plasticity) and are
even more dynamic during development.
One class of the regulatory molecules that we study comprises small families of proteases that activate and inactivate various growth factors,
enzymes and extracellular structural macromolecules.
An example is the BMP1/Tolloid-like family of proteinases (see the Figure, below).
Another class comprises antagonists of signaling by members of the TGFb superfamily of growth factors.
Three additional classes of regulatory molecules under study modify actions of the first two by as yet unclear mechanisms.
Molecules we study play central roles in morphogenetic processes and defects in these proteins may underlie human disease.
Our studies employ biochemical, genetic and developmental analyses, including characterizing standard and conditional "knockout" mice, and
zebrafish and examining possible links between defects in genes of interest and human development and disease.
Current studies in the lab also include roles of extracellular proteins in adipocyte biology, diabetic symptoms,
atherosclerosis, and organ transplant rejection. The latter two studies include translational studies of human patients.
Fig. 1. An overview of the roles of BMP1/Tolloid-like proteinase in morphogenetic events (click on figure for enlarged image).
BMP1/Tolloid-like proteinases process ECM precursors (e.g. procollagen) to mature functional ECM components.
They also activate TGFb1 by processing latent binding protein 1 (LTBP1), thereby releasing truncated
large latent complex (LLC) from the ECM, leading to consequent activation via cleavage of the TGFb1 prodomain, known as latency-associated peptide
(LAP), by non-BMP1/Tolloid-like metalloproteinases (e.g. MMP2). Activated TGFb1 binds cell surface receptors (TGFb-R),
which activate R-Smads 2 and 3, which then combine with Smad4 for translocation to the nucleus, and up-regulation (arrows)
of BMP1, ECM precursors (e.g. procollagen), MMP2, and TGFb1 itself. TGFb1 also down-regulates some MMPs that
degrade ECM (e.g. MMP1). The BMP1/Tolloid-like proteinases thus appear to be involved in a positive feed-back/feed-forward
loop for morphogenetic events/tissue remodeling. BMP1/Tolloid-like proteinases also activate BMPs 2/4 by cleaving
the antagonist Chordin, thus inducing activation of R-Smads 1, 5, and 8. These may compete with R-Smads 2 and 3 for limiting amounts of Smad4.
Chordin might also compete with ECM precursors and LTBP1 for BMP1/Tolloid-like proteinases.
Smad4 and BMP1/Tolloid-like proteinases may therefore represent two levels at which cross-talk between TGFb and BMP
signaling pathways orchestrates tissue remodeling with patterning. (from Hopkins et al., Matrix Biol. 26, 508-523, 2007).
Naturally occurring antagonists of the BMP1/Tolloid-like proteinases include the proteins a2-macroglobulin (a2M) and sizzled.
Other modulators of the activities of the BMP1/Tolloid-like proteins, under study in the lab, are the procollagen C-proteinase enhancers 1 and 2, and sFRP2 (not shown).
Muir AM, Massoudi D, Nguyen N, Keene DR, Lee SJ, Birk DE, Davidson JM, Marinkovich MP, Greenspan DS. (2016) BMP1-like proteinases are essential to the structure and wound healing of skin. Matrix Biol. Jun 27. pii: S0945-053X(16)30142-1. doi: 10.1016/j.matbio.2016.06.004. [Epub ahead of print]
- Park AC, Huang G, Jankowska-Gan E, Massoudi D, Kernien JF, Vignali DA, Sullivan JA, Wilkes DS, Burlingham WJ, Greenspan DS. (2016) Mucosal Administration of Collagen V Ameliorates the Atherosclerotic Plaque Burden by Inducing Interleukin 35-dependent Tolerance. J Biol Chem. 291:3359-70.
- Park AC, Phillips CL, Pfeiffer FM, Roenneburg DA, Kernien JF, Adams SM, Davidson JM, Birk DE, Greenspan DS. (2015) Homozygosity and Heterozygosity for Null Col5a2 Alleles Produce Embryonic Lethality and a Novel Classic Ehlers-Danlos Syndrome-Related Phenotype. Am J Pathol. 185:2000-11.
- Sullivan JA, Jankowska-Gan E, Shi L, Roenneburg D, Hegde S, Greenspan DS, Wilkes DS, Denlinger LC, Burlingham WJ. (2014) Differential requirement for P2X7R function in IL-17 dependent vs. IL-17 independent cellular immune responses. Am J Transplant. 4:1512-22.
- Muir AM, Ren Y, Butz DH, Davis NA, Blank RD, Birk DE, Lee SJ, Rowe D, Feng JQ, Greenspan DS.(2014) Induced ablation of Bmp1 and Tll1 produces osteogenesis imperfecta in mice. Hum Mol Genet. 23:3085-101.
- Vittal R, Fan L, Greenspan DS, Mickler EA, Gopalakrishnan B, Gu H, Benson HL, Zhang C, Burlingham W, Cummings OW, Wilkes DS. (2013) IL-17 induces type V collagen overexpression and EMT via TGF-β-dependent pathways in obliterative bronchiolitis. Am J Physiol Lung Cell Mol Physiol. 304:L401-14.
- Yang C, Park AC, Davis NA, Russell JD, Kim B, Brand DD, Lawrence MJ, Ge Y, Westphall MS, Coon JJ, Greenspan DS. (2012) Comprehensive mass spectrometric mapping of the hydroxylated amino acid residues of the α1(V) collagen chain. J Biol Chem. 287:40598-610.
- Asharani PV, Keupp K, Semler O, Wang W, Li Y, Thiele H, Yigit G, Pohl E, Becker J, Frommolt P, Sonntag C, Altmüller J, Zimmermann K, Greenspan DS, Akarsu NA, Netzer C, Schönau E, Wirth R, Hammerschmidt M, Nürnberg P, Wollnik B, Carney TJ. (2012 Attenuated BMP1 function compromises osteogenesis, leading to bone fragility in humans and zebrafish. Am J Hum Genet. 90:661-74.
- Huang G, Greenspan DS. (2012) ECM roles in the function of metabolic tissues. Trends Endocrinol Metab. 23:16-22.
- Muir A, Greenspan DS. (2011) Metalloproteinases in Drosophila to humans that are central players in developmental processes. J Biol Chem. 286:41905-11.
- Kim B, Huang G., Ho W-B, Greenspan DS. Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3. J. Biol. Chem. 286, 29014-29025 (2011)
- Huang G, Ge G, Wang D, Gopalakrishnan B, Butz DH, Colman RJ, Nagy A, Greenspan DS.
a3(V) collagen is critical for glucose homeostasis due to effects in islets and peripheral tissues in mice. J. Clin. Invest. 121, 769-783 (2011).
- Dart ML, Jankowska-Gan E, Huang G, Roenneburg DA, Keller M R, Torrealba JR, Rhoads A, Marshall-Case S, Kim B, Bobadilla JL, Haynes LD, Wilkes DS, Burlingham WJ, and Greenspan DS. IL-17-dependent autoimmunity to collagen type V in atherosclerosis. Circ. Res. 107, 1106-1116 (2010). PMCID: PMC3010213
- Branam AM, Hoffman GG, Pelegri F, Greenspan DS. Zebrafish Chordin-like and Chordin are functionally redundant in regulating patterning of the dorsoventral axis. Dev. Biol., 341, 444-458 (2010). PMCID: PMC2862114
- Huang G, Zhang Y, Kim B, Ge G, Annis DS, Mosher DF, and Greenspan DS. Fibronectin binds and enhances the activity of bone morphogenetic protein 1. J. Biol. Chem. 284, 25879-25888 (2009). PMCID: PMC2757989
- Kobayashi, K, Luo M, Zhang Y, Wilkes DC, Gao G, Grieskamp T, Yamada C, Liu T-C, Huang G, Basson CT, Kispert A, Greenspan DS*, Sato, TN*. Secreted Frizzled Related Protein 2 is a procollagen C-proteinase enhancer with a role in fibrosis associated with myocardial infarction. Nature Cell Biol. 11, 46-55 (2009) (*Co-contributing authors). PMCID: PMC2722759
- Ge G, Fernandez C A, Moses MA, Greenspan DS. Bone morphogenetic protein 1 processes prolactin to a 17-kDa anti-angiogenic factor. Proc. Natl. Acad. Sci. U. S. A. 104, 10010-10015 (2007). PMCID: PMC1891225
- Jasuja R, Ge G, Voss NG, Lyman-Gengerich J, Branam AM, Pelegri FJ, Greenspan DS. Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling of bone morphogenetic proteins 2 and 4. J. Biol. Chem. 282, 9053-9062 (2007)
- Ge G, Greenspan DS. BMP1 Controls TGFb1 Activation Via Cleavage of Latent TGFb-binding Protein. J. Cell Biol. 174, 111-120 (2006). PMCID: PMC2064503
- Steiglitz BM, Kreider JM, Frankenburg EP, Pappano WN, Hoffman GG, Meganck JA, Liang X, Hook M, Birk DE, Goldstein SA, Greenspan DS. Procollagen C-proteinase enhancer 1 genes are important determinants of the mechanical properties and geometry of bone and the ultrastructure of connective tissues. Mol. Cell. Biol. 238-249 (2006) PMCID: PMC1317636
- Ge G, Hopkins DR, Ho W -B, Greenspan DS. GDF11 forms a BMP1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells. Mol. Cell. Biol. 25, 5846-5858 (2005) PMCID: PMC1168807
- Pappano WN, Steiglitz BM, Scott IC, Keene DR, Greenspan DS. Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of BMP-1/Tolloid-like metalloproteinases. Mol. Cell. Biol. 23, 4428-4438 (2003) PMCID: PMC164836
- Scott IC, Blitz IL, Pappano WN, Maas SA, Cho KWY, Greenspan DS. Twisted gastrulation homologs are extracellular cofactors in antagonism of BMP signaling. Nature 410, 475-478 (2001)
- Clark TG, Conway SJ, Scott IC, Labosky PA, Winnier G, Bundy J, Hogan BLM, Greenspan DS. The mammalian tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart. Development 126, 2631-2642 (1999)
- Toriello HV, Glover TW, Takahara K, Byers P, Miller DE, Higgins JV, Greenspan DS. A translocation interrupts the COL5A1 gene in a patient with Ehlers-Danlos syndrome and hypomelanosis of Ito. Nature Genet. 13, 361-365 (1996)
- Kessler E, Takahara K, Biniaminov L, Brusel M, Greenspan DS. Bone Morphogenetic Protein-1: the type I procollagen C-proteinase. Science 271, 360-362 (1996) (See Perspectives Science 271, 463)