Regulating Mechanism Of A Novel Bone Absorption Regulator OCIL And Its Action On The Development Of Osteoclasts

Osteoclast inhibitory lectin (OCIL), a recently identified potent inhibitor of osteoclast formation. Structurally, OCIL belongs to type II transmembrane molecules with a C-type lectin extracellular domain. It has similar tissue distribution to that of RANKL/OPG system. OCIL dose-dependently inhibited multinucleate osteoclast formation from adherent murine spleen cells treated with RANKL and M-CSF; But it can not neutralize the effect of RANKL on osteoclastogenesis. These evidences strongly suggested that OCIL might have a direct action to oppose RANKL in the control of osteoclastogenesis. In view of the strong inhibiting effect of OCIL on osteoclastogensis, scientists predict that OCIL may be developed to the preventive drug of osteoporosis. However, little information is available to date concerning OCIL actions. Furthermore a variety of osteotropic factors in osteoblastic cells, including retinoic acid,1,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, interleukin-la and interleukin-11upregulate OCIL mRNA expression in osteoblast-like cells. But the precise mechanism on how the expression of this gene is controlled in osteoblast-like cells remains to be clarified.To make it clear, we explored how OCIL is controlled in osteoblast; and we also carry out the researches on "the molecular mechanism of inhibiting effect of OCIL on osteoclastogensis",In first part we focused on whether PTHrp(1-34) regulates OCIL expression and the signaling pathways used. Using real-time RT-PCR, we demonstrated in rat osteoblast-like UMR-106cells, PTHrp(1-34) caused a concentration-and time-dependent increase in OCIL expression that began and reached maximum later than RANKL induction and OPG suppression. In murine MC3T3-E1cells, PTHrp(1-34) also elicited a slight increase of OCIL at the protein level as evidenced by Western blotting analysis.After that the intracellular signal transduction pathways involved in mediating the effect of PTHrp(1-34) on the expression of OCIL mRNA in UMR106osteoblast like cells was investigated. The enhancement in OCIL mRNA in UMR-106cells was abolished by RNA synthesis inhibitor actinomycin D. Nevertheless, protein synthesis inhibitor cycloheximide dramatically induced OCIL expression, which was not superinduced by the cotreatment with PTHrp(1-34). We also confirmed that cAMP/PKA signaling activators PTH(1-31), forskolin and dibutyryl cAMP (db-cAMP) all increased OCIL mRNA levels. Calcium ionophore A23187also dramatically induced OCIL expression. In contrast, phorbol-12-myristate-13-acetate (PMA) that stimulates PKC rather than PKA, reduced OCIL expression in short term but induced OCIL mRNA in long term. Selective PKA inhibitor KT5720, mitogen-activated protein kinase (MAPK) cascade inhibitor PD98059, calmodulin antagonist W-7and Ca+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) inhibitor KN-62all significantly blunted PTHrp-stimulated OCIL expression. On the other hand, PTHrp(1-34) induced OCIL mRNA expression was markedly increased by PKC-specific inhibitor chelerythrine Moreover, PD98059could block the stimulation of OCIL by db-cAMP but not that by A23187. Collectively, these data established that PTHrp(1-34) regulates OCIL expression in vitro, cAMP/PKA, Ca2+/CaMK Ⅱ and MAPKsignaling pathways mediate the induction of OCIL.In part two, to further investigate how expression of OCIL is regulated in osteoblastic cells and identify the regulatory elements controlling OCIL gene expression, approximately4.5kb of the5’-flanking sequence of rat OCIL gene was placed upstream of the luciferase reporter gene pGL3-Basic and transiently transfected into three different cells that differed in their expression of the endogenous OCIL gene. The differences in the levels of are quite consistent with the differences in the levels of OCIL expression in these cells, suggesting that the regulation of rat OCIL gene expression occurs mainly at the transcriptional level. Upon these findings, luciferase reporter vectors harbouring various lengths of the rat OCIL5’flanking regions were constructed in the pGL3-basic luciferase reporter vector and luciferase activity were analyzed with transient transfection experiments. The results revealed that these5’flanking deletion constructs of OCIL promoter demonstrated different level of luciferase activity. On the basis of these findings, the minimal promoter and some regions that may have important regulatory elements were identified. In summary, this work represents a preliminary but crucial step towards the identification of the PTHrp responsive region of OCIL promoter and core elements that contribute to the stimulatory effect of PTHrp. It also provides a very useful and convenient mean for further studying the regulatory mechanism of OCIL by PTHrp and other regulatory factors of bone metabolism as well.In part three,the roles of OCIL gene were investigated in regulating the mRNA expression of TRAP、MMP-9and CTSK gene and in affecting osteoclastogenesis. First to construct a CHO cell line secretory expressing mOCIL, a cDNA fragment encoding mOCIL gene was amplified by PCR from pMal/mOCIL plasmid which contains the full cDNA sequence of mOCIL. Then we spliced the chemical synthesized peptide sequence of IgK+and mOCIL fragment into a fusion gene by using overlap extension PCR technique.Subsequently we inserted sig-mOCIL into a eukaryotic expression vector pcDNA3.1and then the vector was transfected into CHO cells with lipofectaMINE2000. Under the pressure of G418,we screened out cells that expressed the mOCIL gene permenantly.Then the number of osteoclast and the mRNA levels of TRAP、MMP-9and CTSK in osteoclasts were investigated. We found that the number of osteoclast and the mRNA levels of TRAP、MMP-9and CTSK in osteoclasts decreased significantly when we used the medium of CHO cells transfected with the fusion gene sig-mOCIL to interfere with primary murine bone marrow cells induced by PTHrp. It indicates that OCIL gene may influence the differentiation and activity of osteoclast by inhibiting the expression of TRAP、 MMP-9、CTSK mRNA. This research establish a foundation to the further study on the molecular mechanism of how OCIL gene influence the development and function of osteoclast.