The Role Of Integrin β1 In Cytokine-induced Osteoclast Differentiation

Bone tissue changes are largely regulated by the combined action of two bone cell types-osteoblasts and osteoclasts.Osteoblasts are primarily responsible for new bone formation,while osteoclasts are responsible for removing mineralized tissue.Their formation and activity are tightly regulated by a combinatorial action of multiple hormones and cytokines within the bone microenvironment.Disturbance in these cellsaltered mass and activity underlies many pathological bone conditions.In particular,most adult skeletal disease such as osteoarthritis,heumatoid arthritis is related to abnormal osteoclast activity.Proinflammatory secretions such as TNF-a,IL-6 produced in the pathological bone tissues have been implicated to progressively modulate the osteoclast activity,facilitating the disease progression,and hence targeting their involvement has been suggested as an intervention to reduce bone erosion.Studies from targeted knockout mutations or other naturally occurring mutations have identified at least 24 gene loci to regulate Osteoclast differentiation and activation.However,recent advance have underscored the importance of the tumor necrosis factor(TNF)receptor(TNFR)/TNF-like proteins:(a)osteoprotegerin(OPG),(b)receptor activator of nuclear factor(NF)-κB(RANK)and(c)RANK ligand(RANKL)to regulate osteoclast function.It is now known that RANKL or TNF-αcombined with macrophage colony stimulating factor(MCSF)alone is sufficient to induce osteoclastogenesis from bone marrow macrophage cells in vitro.However,deeper understanding of how diverse physiological and pathological signals modulate the RANKL or TNF-α pathway and osteoclast functions would allow intervening therapeutics to regulate bone erosion.Integrins are heterodimeric receptors mediating cell-cell and cell-matrix interactions and a role for them has been widely appreciated in the osteoclast differentiation and activation.Expression analyses of osteoclast have identified at least three major integrin types-alpha V beta 3(α5β3),alpha 5 betal(α5β1),alpha 2 beta1(α2β1).Although a critical role for α5β3 integrin has been defined in osteoclastogenesis,the other integrin subtypes remain largely unexplored.Given that most of them involve integrin-β1,understanding the functions through integrin-β1 would allow understanding the functions of both the integrin subtypes and other functions involving integrin-β1 in osteoclasts.In this study,the role of integrin subtype integrin-β1 has been highlighted to specifically regulate the TNF-α induced osteoclast formation and activation by modulating MAPK signaling.In addition,blocking this pathway through integrinβ1 blocking antibody or MAPK inhibitor showed similar results and thereby provide a viable route for therapeutic intervention.The shRNA sequences targeted for integrin-β1 were adapted from vendor sources(validated clones from Sigma RNAi consortium)and also previously used and validated elsewhere.shRNA sequences were cloned into lentiviral vector(pLKO.1)and infected in RAW264.7.The cells were then selected with puromycin and evaluated for knockdown efficiency.Cellstransduced with empty vector showed similar integrin-β1 expression levels as normalcells,whereas cells transduced with shRNA sequences had varying levels of reduction.Cells with the greatest reduction of integrin-β1(-85%reduction)were designated as integrin-β1 knock down cells and used in subsequent experiments to mimic the loss of gene function phenotype.To evaluate whether integrin-β1 is essential for osteoclastdifferentiation,we examined the effects of loss of integrin-β1 function in vitroosteoclast differentiation.Stable knockdown of integrin-β1 strongly decreased the formation of TRAP positive osteoclasts in TNF-α induced osteoclastogenesis,while RANKL treated cells differentiated normally.These observations were further evaluated for functional significance through pit formation assay.In accordance with the osteoclast formation trends,integrin-β1 knockdown cells showed reduced pit formation upon TNF-α treatments and no difference were seen in the RANKL treatments.Finally,the results were confirmed by the decreased expression of osteoclast differentiation marker genes such as Osterix and Cathespin k in integrin-β1 knockdown cells in TNF-α treatments,and no change in RANKL treatments were observed.Together,these results suggest that the integrinβ1 related signaling plays a critical role in TNF-α induced osteoclast differentiation.To elucidate the molecular mechanisms relating integrin-β1 with osteoclastogenesis,we evaluated for changes in signaling cascades associated with osteoclast differentiation.We compared the changes between control and integrin-β1 knockdown cells upon treatment with either RANKL or TNF-α.In response to TNF-α stimulation,loss of integrin-β1 showed reduced p38 MAP kinase phosphorylation,while no differences were seen in the phosphorylation status of JNK signaling,and IκB levels between control and integrin-β1 knockdown cells.In contrast,upon RANKL stimulation no significant changes were seen between control and integrin-β1 knockdown cells in any of the signaling pathways investigated including p38.However,all the pathways investigated showed increased response to RANKL.To further confirm the specificity of Integrin-β1 and p38 MAP kinase signaling in relation to osteoclast differentiation,the integrin-β1 knockdown cells were treated with p38 MAP kinase inhibitor and an augmented reduction in osteoclast differentiation were observed in integrin-β1 knockdown cells only in TNF-α treatments.Surprisingly,though increased p38 response was seen in RANKL treatment,p38 inhibitor does not show a significant reduction in osteoclast differentiation.Although speculative,this implies that the significance of MAPK kinase signaling is minimal in RANKL induced osteoclasts,which is in contrast to TNF-α induced osteoclast formation.Together,this data confirms that integrin-β1 is critical and functions via MAP kinase signaling in TNF-αinduced osteoclast differentiation.Based on the observation for a role for integrin-β1 in mediating osteoclast differentiation,we further investigated whether the integrin-β1 blocking antibodies could modulate the signaling and osteoclast formation.When control cells were continuously treated with increasing doses of integrin-β1 blocking antibody,the formation of TRAP multinucleated cells upon TNF-α treatment was attenuated in a dose dependent manner,while no significant changes were seen in RANKL treated cells.Similar results were observed upon MAPK kinase inhibitor treatments in TNF-α treatments and no change was seen in RANKL treated cells.Overall,the data presented in this study underscores the critical role of integrin-β1 in TNF-α-induced osteoclast formation and resorption,and its potential to reducepathological osteolysis.Future studies validating the role of integrin-β1 in osteoclast maturation and activation from in vivo pathological bone tissues,and improvements in developing efficient antibody-based therapeutic options or other alternate routes to target the pathway could translate into a viable management option for chronic bone disease.