CD735’-ecto-nucleotidase acts as a regulatory factor in osteo-chondrogenic differentiation of mechanically stimulated mesenchym.docx

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1、A Ode et al.CD73 controls differentiation ofMSCsEuropean A Ode et Cells”/, and Materials Vol. 25 2013 (pages37-47)CD735,-ECTO-NUCLEOTIDASE ACTS AS A REGULATORY FACTOR INOSTEO-CHONDROGENIC DIFFERENTIATION OF MECHANICALLYSTIMULATED MESENCHYMAL STROMAL CELLSAndrea Ode *, Janosch Schoon , nnctt Kurtz ,

2、Marcel Gactjcn , Jan E. Ode , Sven Geissler , Georg N. Duda1.21,2111L21.2,Julius Wolff Institute and Musculoskeletal Research Center Berlin, Charitc-Univcrsitatsmcdzin, Berlin, Germany2 Berlin-Brandenburg Center fbr Regenerative Therapies, Berlin, GermanyAbstractIntroduction38www.ecnjournaLorgBone r

3、egeneration is influenced by mesenchymal stromalcells (MSCs) and mechanical conditions. How healingoutcome and mechanical stability arc linked on the cellularlevel, however, remains elusive. Cyclic-comprcssivcloading of MSCs accts the expression of moleculesinvolved in angiogenesis and matrix assemb

4、ly, but alsoreduces the expression of CD73, an ccto-5-nucleotidase,which plays a crucial role in extracellular adenosinegeneration. Although, fbr almost 20 years, CD73 has beena major cell surface marker defining MSCs, little is knownabout its function in these cells. Therefore, the aim of thisstudy

5、 was to determine the putative involvement of CD73in MSC differentiation after cyclic-comprcssivc loading.After cultivation in appropriate differentiation media,chondrogcnic differentiation ability was significantlyincreased in loaded MSCs, hence following current models.Through treatment with the C

6、D73 inhibitor adenosine5-(, -methylene) diphosphate, chondrogenic matrixdeposition was further increased; in contrast, mineralmatrix deposition and expression of osteogenic markers wasreduced. One major signal transduction pathway, which isactivated via CD73-mcdiatcd adenosine, is the adenosinerecep

7、tor pathway. Thus, the adenosine receptor expressionpattern was investigated. MSCs expressed the four knownadenosine receptors at the mRNA level. After mechanicalstimulation ofMSCs, Adora2a was down-regulated. Thesedata point towards a role of CD73 in MSC differentiationpossibly vA2R signalling, whi

8、ch is mutually regulatedwith CD73. In conclusion, the findings of this studysuggest that CD73 is another regulatory factor in ostco-chondrogcnic differentiation of MSCs and may providea - thus far underestimated - therapeutic target to guidebone regeneration.Keywords: Mesenchymal stromal cells; mech

9、anicalstimulation; CD73/5-ccto-nuclcotidasc; chondrogcnicdifferentiation; osteogenic differentiation.* Address fbr correspondence:Andrea Ode,Julius Wolff Institute and Center fbr MusculoskeletalSurgery,Charite-Universitatsmedizin Berlin.Bone healing is a complex, however well-orchestrated,multistage

10、 regenerative process. Bone fracture coincideswith disruption of blood vessels resulting in activation ofthe coagulation cascade and formation of the haematoma,which encloses the fracture area. Inflammatory cells,fibroblasts, and mesenchymal stromal cells (MSCs) arcrecruited to the site. Once MSCs h

11、ave reached the bonefracture site they arc confronted with a challenging milieucharacterised not only by inflammatory cytokines and lowoxygen tension (hypoxia) but also by constant mechanicalstrain (Goodship and Kcnwright, 1985; Komatsu andHadjiargyrou, 2004). This condition is likely to affectMSCs,

12、 which arc known to be mcchanoscnsitivc (Wangand Thampatty, 2008). Thus, detailed knowledge aboutthe influence of mechanical loading on MSCs is pivotalfbr understanding the physiological processes during boneregeneration in order to develop innovative cell therapyapproaches.Recently, vve and others

13、provided evidence thatmechanical strain leads to reduced expression of the cellsurface marker CD73 in vitro (Kang et al., 2011; Ode et al.2011). In our study, bone marrow (BM)-MSCs underwentcyclic-comprcssivc loading fbr three days. CD73 proteinand mRN expression were significantly reduced. Ofnote,

14、this cHcct persisted fbr a week after removal ofthe loading stimulus (Ode et al., 2011). In another study,umbilical cord-derived MSCs underwent cyclic uniaxialstretching fbr ten days. Here, the expression of CD73was also significantly decreased as strain increased(Kang et al., 2011). CD73 is an ccto

15、-5,-nucleotidase thatis attached to the outer plasma membrane by a glycosylphosphatidylinositol (GPI)-anchor. It catalyses thehydrolysis of the phosphoric ester bond of 5-ribo- anddcoxyribonuclcotidcs to the corresponding ribo- anddeoxyribonucleoside, and phosphate (Hunsucker etal. 2005; Stratcr, 2006), c.g. it generates extracellularadenosine by the dephosphorylation of adenosine5-monophosphate. Although CD73 is one majorcell surface marker defining MSCs according to TheInternational Society fbr Cellular Therapy (ISCT), it issurprising that little is known about the fnc