Reference Number: 120115
Principal Investigator: Dr. Marco Maccarana
Organization: Lund University
Project Period: April, 2012 – March, 2013
Grant Title: Functions of dermatan sulfate investigated in mice with targeted
DS-epimerases
Abstract
Chondrotin/dermatan sulfate (CS/DS) is a polysaccharide which is present, sometimes aspredominant component, virtually in all types of extracellular matrices and on the surface of
all cells. The polysaccharide CS/DS contains oligosaccharide domains with exquisite
structural and biological properties. CS is converted into DS by epimerization of glucuronic
acid into iduronic acid (IdoA). Three enzymes are specifically involved in dermatan sulfate
formation: dermatan sulfate epimerase 1 and 2 (DS-epi1 and 2) and dermatan sulfate
4-O-sulfotransferase 1 (D4ST1). Our group has cloned DS-epi1 and 2, established the
deficient mouse models and is in the position to investigate these models to elucidate in vivo
functions of dermatan sulfate.
Objectives
The objective was to increase the understanding of the functions of the DS-epimerases bycharacterizing the DS-epi2-/- mice (5), to identify the role of DS-epi1 in esophagus squamouscell carcinoma (3) and its role in migration of aortic smooth muscle cells (1).
Methods used
Iduronic acid analysis. Brain extracellular matrix investigation by IHC (5). Mass
spectrometric structural analysis of CS/DS on human biopsies. Migration and invasion
cellular assays. Biochemical analysis of cellular signaling events (3). Cellular migration
recorded in living cells. Confocal and total internal reflection fluorescence microscopy (1).
Results
DS-epi2 deficient mice were generated (5). While DS-epi1 is the major epimerase in mosttissues, in kidney and brain DS-epi2 is responsible for approximately 50% and 90% of thetotal epimerase activity, respectively. The last two tissues were examined: despite its highexpression, the structure of CS/DS in DS-epi2-/- kidney is unchanged and in DS-epi2-/- brain
there is a mere 30% reduction of iduronic acid. The content and distribution of CS/DS inbrain was not affected, neither the general organization of the extracellular matrix.
DS-epi2-ablated mice with the mixed C57BL/6-129/SvJ genetic background were born at the
expected Mendelian frequency, were vital and fertile, and no obvious anatomical andhistological abnormalities were observed.
The functions of DS-epi1 were investigated in two contexts: one was cancer, and more
precisely human esophageal squamous cell carcinoma (3), and the second was the effect on
cellular motility, in particular in aortic smooth muscle cells (1).
DS-epi1 is highly expressed in squamous cell carcinoma, both in the stroma and in the cancer
cells. The fine structure of CS/DS in human biopsies was examined and revealed differences,also in iduronic acid content, between cancer and normal tissue. In cells derived from humanesophageal squamous cell carcinoma downregulation of DS-epi1 allowed us to recognize theimportance of IdoA in hepatocyte growth factor-mediated cellular motility and invasion.
Aortic smooth muscle cells from wild type and DS-epi1-/- mouse differed in their ability torepopulate wounded areas due to loss of directional persistence of migration. DS-epi1-/- cells,
however, had not lost the general property of migration showing even increased speed ofmovement compared to wild type cells. This could be explained by a decrease of focal
adhesion sites and expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK).