| Background: The intervertebral disc(IVD) consists of three basic structures: the central gel-like nucleus pulposus(NP), the outer annulus fibrosus(AF), and the superior and inferior cartilage endplates. The IVD is the primary load ‐bearing tissue that permits flexion as well as rotation and extension of the human spine. The extracellular matrix secreted by NP cells play a most important role in maintaining intervertebral disc physiological function. The NP cells can secrete a complex extracellular matrix that contains proteoglycans, such as Aggrecan, and various types of collagen, and proteoglycan content is the highest, accounted for over 50% of dry weight of NP tissue. Therefore, the degradation of proteoglycans is one of the important factors in intervertebral disc degeneration. ADAMTS-4 and ADAMTS-5, the members of the ADAMTS(A Disintegrin and Metalloproteinase with Thrombospondin motifs,) family is currently recognized as the most important aggrecanases. Previous studies have confirmed that the the cytokines in microenvironment of degenerated intervertebral disc, such as TNF-α, IL-1β and so on, stimulate ADAMTS-4 and ADAMTS-5 high expression in the NP cells, while degradation of aggrecan is increased. Thus, it could be suggest plays an important role in the process of intervertebral disc degeneration. Hence the correlative study of ADAMTS-4 and ADAMTS-5 will enable us to make a further understanding about the molecular mechanism of intervertebral disc degeneration.Nuclear factor of activated T-cells(NFAT) family consist of NFAT1-4 and NFAT5. NFAT1-4 were regulated by calcium signaling pathways. NFAT5 also called osmotic response element binding protein(OREBP) or tonicity-responsive enhancer binding protein(Ton EBP). And NFAT5 was only transcripition factor respond to hypertonicity environment in mammal. Previous studies have been shown that calcium signaling pathways play an important role in aggrecan synthesis and contribute to IVD bear load. As we all know, microenvironment of IVD was hypertonic. Previous view suggests that microenvironment of NP cells was unfavourable to metabolic reactions, but recent studies had shown that hypertonic environment of NP cells may promote the synthesis of extracellular matrix, such as aggrecan, through activated Ton EBP. Relevant studies mainly focus on some of the anabolic factors, however, the role of hyperosmosis to key aggrecanases, ADAMTS-4 and ADAMTS-5, has not yet been studied. This experiment will first study the regulation mechanism of ADAMTS-4 and ADAMTS-5 expression in Response to calcium and hyperosmosis, and role of NFAT in this process.Objective: The objective of the investigation was to study underlying regulation mechanism of ADAMTS-4 and ADAMTS-5 expression in Response to calcium and hyperosmosis, and to explore the potential signal transduction pathways involved in the regulation of ADAMTS-4 and ADAMTS-5 with calcium and hyperosmosis(NFAT).Methods: Rats nucleus pulposus cells were cultured in vitro, the NP cells were treated with the medium that was adjusted osmotic pressuring with 10 m M Na Cl solution(330osm,450 osm,550osm) for 24 hours. Then,We examine the level of ADAMTS-4 and ADAMTS-5 m RNA and protein in conditioned medium of treated NP cells by realtime RT-PCR,Western blot analysis. To measured regulation of ADAMTS-4 and ADAMTS-5 promoter activity in response to hyperosmosis, we transfected NP cells with PGL3-ADAMTS4 or PGL3-ADAMTS5 reporter gene plasmids. And we investigated the expression of Ton EBP in NP cells treated with hypertonic medium by Western blot; To confirm the role of Ton EBP, we used the JASPAR database analysis for evidence of putative binding motifs of NFAT family in ADAMTS-4 and ADAMTS-5 promoter. Then, To detect the role of Ton EBP, we transfected NP cells with Ton EBP plasmids with ADAMTS-4 or ADAMTS-5 promoter plasmids. To further conform the role of Ton EBP in hyperosmosis regulated ADAMTS-4 and ADAMTS-5, we treated NP cells with hypertonic medium after co-transfected with dominant-negative Ton EBP(DN-Ton EBP) plasmids and ADAMTS-4 or ADAMTS-5 promoter plasmids. To confirm role of calcium, after treated with ionomycin and PMA, ADAMTS-4 and ADAMTS-5 m RNA were examined by real-time RT-PCR. To investigate role of calcium and calcium signaling pathways, NP cells were treated with BAPTA-AM or Cs A and FK506, ADAMTS-4 and ADAMTS-5 promoter activity were detected by dual-luciferase system. Moreover, we cotransfected NP cells with NFAF1,2,3,4 expression plasmids and ADAMTS-4 or ADAMTS-5 promoter plasmids, to confirm the regulation of NFAT family on ADAMTS-4 and ADAMTS-5 promoter activity.Results: The result of real-time RT-PCR, western blot analysis show that hyperosmosis can inhibit expression of ADAMTS-4 and ADAMTS-5 in m RNA and protein. We successfully constructed PGL3-ADAMTS-4and PGL3-ADAMTS-5 reporter gene plasmids, and we used the JASPAR database analysis for ADAMTS-4 and ADAMTS-5 promoter sequences, it show 2 putative binding motifs of NFAT family in ADAMTS-4promoter and 3 putative binding motifs of NFAT family in ADAMTS-5 promoter. Western blot results show that the hyperosmosis can increase Ton EBP expression. To ascertain if the hyperosmosis inhibit expression of hyperosmosis through Ton EBP, cell treated with hypertonic medium after co-transfected with DN-Ton EBP and ADAMTS-4 or ADAMTS-5 promoter plasmids, it showed DN-Ton EBP had no effect on suppression of ADAMTS-4 expression, but DN-Ton EBP relieved suppression of ADAMTS-5 expression. Similarly, NP cells transfected with Ton EBP and ADAMTS-4 or ADAMTS-5 promoter plasmids, it showed Ton EBP had no effect on ADAMTS-4 expression, but Ton EBP showed a robust suppression on ADAMTS-5 expression. ADAMTS-4 and ADAMTS-5 m RNA increased after NP cells treated with ionomycin and PMA. Inductive effect of ADAMTS-4 and ADAMTS-5 m RNA were blocked by NP cells treated with BAPTA-AM or Cs A and FK506. Moreover, NFAT-1, show a suppression on ADAMTS-4, but a induction on ADAMTS-5, both NFAT-2 and NFAT-3 increase the expression of ADAMTS-4 and ADAMTS-5, NFAT-4 showed no effect on ADAMTS-4 and ADAMTS-5.Conclusion: 1. The high osmotic pressure i has inhibitory effect on ADAMTS-4 and ADAMTS-5 and the inhibitory effect on the physiological concentration range showed a dose dependent. 2.In this procedure, Ton EBP associate with ADAMTS-5, but not to ADAMTS-4. 3. Expression of ADAMTS-4 and ADAMTS-5 increased by calcium and calcium signaling pathwats. 4. promoter region of ADAMTS-4 and ADAMTS-5 may have putative binding motifs of NFAT family, and NFAT-1show the suppressive effect on ADAMTS-4, but a inductive effect on ADAMTS-5. Both NFAT-2 and NFAT-3 show the significant increase, NFAT-4 show no effect on both 2 genes. |
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