The Study Of The Biological Role And The Underlying Mechanism Of CGRP In Bone Restoration And Inflammatory Bone Disease

The unique capacity of restoration and regeneration of bone owe to a series of cells and molecules which regulate the development of bone. Osteocytes behavior play an important role in the process of bone healing. In fact, the maintenance of bone homeostasis is under strict control, which depend on the coupling between bone resorption and bone formation resulting from the cellular signal communication between osteoblasts and osteoclasts. This close coupling is vital for the physiological function of skeletal system.The main function of the skeletal system include: support of the physical structure, regulation of hematopoietic system. The process of remodeling and reconstruction in bone is closely associated with the osteoblasts-caused bone formation and the osteoclasts-caused bone resorption. This process is regulated by the development and activation of these cell types and is mediated by a complex signaling network.Osteoblasts, derived from mesoderm progenitor cells, will produce bone matrix when they are fully differentiated. This highly developed cell lines, is the main functional cells in the bone formation metabolism. Many signaling pathways, such as ERK pathway, OPG/RANKL pathway, BMP-2/Smads pathway and Wnt pathway, play vital roles in osteoblast differentiation and proliferation. Recently, neurotransmitters have gained a growing interest as the regulator of osteoblasts. Efferent nerves of sympathetic nerves and parasympathetic nerves Among these, the wide-spread CGRP has caused great attention. The sensory neuropeptide substance CGRP, which exists in the cytoplasm in the form of particles, is widely spread in bone tissue, especially in the active site of bone growth. In addition, osteoblastitself can also secrete CGRP.Further evidence comes from the spliced calcitonin gene CGRP, which plays an important role in bone metabolism. It can not only through direct inhibition of osteoclast activation leading to inhibition of bone resorption or indirectly by regulating cytokines released by osteoblasts, such as interleukin-1 and tumor necrosis factor alpha, to influence osteocytes function. In vivo tests show that CGRP can increase the density of bone in transgenic mice. m RNA of CT, CGRP and their receptors are expressed in various osteoblast cell lines and normal human osteoblast-like cells. In these cells, CGRP promotes the cell proliferation and the expression of insulin growth factor.CGRP stimulates the production and activation of protein kinase A(PKA) by stimulating the c-AMP activity and phospholipase through their relevant receptor, and increases the level of intracellular free calcium and the activation of protein kinase. It has been shown that activated protein kinases are involved in CGRP induced proliferation of osteoblasts.In recent years, the immune and nervous system show a widespread neurotransmitter mediated two-way communication, sensory nerve fiber of type C activation can also regulate the immune response and release gene related peptide(CGRP) by neuropeptides such as calcitonin hormone.CGRP receptors are composed of seven transmembrane domain proteins, namely, the calcitonin receptor like receptor(CRLR), the accessory protein and the receptor activity modifying protein(RAMP1). In addition, the CGRP receptor component protein(RCP), a cytosolic protein, appears to be the signal needed for effective signal transduction. And CGRP is the Trimer protein that activates this receptor.In this study, we pay attention to the CGRP receptor mediated function of osteogenic differentiation, the related mechanism underlying the role of bone restoration of CGRP, and the protective effect of CGRP in inflammatory bone disease, with a view to lay the foundation for clinical application of CGRP in bone wound healing.To study the CGRP receptor mediated osteogenic differentiation, we firstly use lentivirus to downregulate the expression of CGRP receptor, RAMP1. The knockdown effect was validated by q PCR, WB, and immunofluorescence. The results showed that RAMP1 knockdown could inhibit the CRLR expression and the cell membrane distribution in osteoblasts. In addition, the stable knockdown of RAMP1 inhibited CGRP-induced cell proliferation and alkaline phosphatase activity, reduced the m RNA expression of Runx2 and type I collagen as well as mineralization deposition. We also found that RAMP1 knockdown could inhibit CGRP-stimulated c-AMP level in osteoblasts.We also validated the protective effect of CGRP on the rat model of periodontitis. The results showed that LPS could induce the apoptosis of osteoblasts, and enhance the expression of p-ERK and cleaved caspase-3. And the treatment with exogenous CGRP exogenous could almost completely inhibit these changes induced by LPS.Methods:(1).The effect of CGRP on the growth in osteoblasts was examined by using the CCK8 assay.When MG63 cells were treated with different concentrations CGRP for 1,4, and 7 days, CGRP significantly increased the cell growth of osteoblasts compared with the control group(P<0.05) in a dose- and time- dependent manner and with a maximal effect at 10-8 M. The ALP activity assay in MG63 cells was also performed. From the results, we could find that CGRP also promoted ALP activity in the osteoblasts in a dose- and time- dependent manner. And the maximal stimulation was observed in the 10-7M CGRP treatment group at the time point of 7 day. Mineralized nodules were investigated by Alizarin Red staining after incubating MG63 cells with osteogenic induction medium and different doses of CGRP for 1,4, and 7 days. Osteoblasts itself had high mineralization potential. During the CGRP-stimulated mineralization process, it still could be obviously observed that CGRP could promote MG63 cells mineralization at both 10-10 to 10-7 M. In short, all of these results indicated that CGRP is a potent activator for osteoblasts which was consistent with previous reports.(2).RAMP1 has now been demonstrated to influence the interaction between CGRP and CRLR, so we try to investigate the influence of knockdown RAMP1 on the expression of CGRP receptors. For this purpose, we used stablely transfected RAMP1-sh RNA osteoblasts to detect the change of other CGRP receptors expression and CGRP distribution. The knockdown of RAMP1 was confirmed by Q-PCR and Western blot. Moreover, the results also showed that osteoblasts in RAMP1-sh RNA treated cells exhibited lower CRLR m RNA and protein expression compared with the cells transfected with Con-sh RNA. Simultaneously, no significant change of RCP expression was observed at either the m RNA or protein level. Next, to identify the expression and the cell membrane localization of CRLR and RCP, immunofluorescent staining were performed. It could be observed that the CRLR staining on the cell membranes in sh RAMP1 transfected osteoblasts was less than that in the Con-sh RNA transfected cells. Meanwhile, the red fluorescence staining representing RCP showed no obvious changes. These results suggested that RAMP1 is important for receptor expression of CGRP and therefore may contribute to CGRP signaling.(3).We speculated that RAMP1 was involved in the stimulation of osteoblast function after exogenous CGRP administration, the proliferation and differentiation in osteoblasts should be inhibited when RAMP1 was knockdown. Consistent with our previous hypothesis, RAMP1-sh RNA treated cells showed a significant reduce of cell proliferation compared to that treated with the control sh RNA.We next examined whether knockdown of RAMP1 would result in inhibition of osteoblast differentiation. Knockdown of RAMP1 aslo inhibited ALP activity of osteoblasts. Furthermore, it could also be observed that the expression of osteoblast differentiation markers RUNX2 and Collagen I significantly decreased in the RAMP1 sh RNA treated cells compared with control sh RNA treated cells. These results suggested that knockdown of RAMP1 would damage the function of osteoblast including proliferation and differentiation after administration of CGRP.(4). The c AMP levels was declined in RAMP1-sh RNA treated cells compared to that in the Con-sh RNA treated cells,this result demonstrated that RAMP1 might be involved with the expression level of CGRP-induced c AMP. Next, the results of CCK8 indicated that cell growth exhibited no obvious changes between RAMP1-sh RNA treated cells and that in the Con-sh RNA treated cells. These data indicated that c AMP/PKA pathway did not influence the RAMP1 knockdown-induced inhibition of proliferation. Next, the effect of RAMP1 knockdown on CGRP induced osteoblastic differentiation was also examined. We observed that the inhibition of ALP activity in RAMP1-sh RNA treated cells could be reversed after forskolin treatment. Similar to the ALP activity,the results exhibited the same trend in m RNA levels of osteoblast differentiation markers. What was more, decreased mineralization degree was also rescued in knockdown cells after treatment of forskolin.(5).To investigate the effect of LPS on the viability of MC3T3-E1 cells, the cells were treated with various concentrations(0-1000ng/ml) of LPS for 48 h. The viability of MC3T3-E1 cells were significantly inhibited by LPS in a dose-dependent manner at concentrations less than 50ng/ml. And there was no significant difference between 500 ng/ml group and 1000 ng/ml group in inhibition of viability. Next, 500 ng/ml LPS treatment for various time periods(0-72h) were performed, the result showed that, after 12 h, as the stimulation time increased, the activity of the MC3T3-E1 cells decreased significantly(P <0.01).Further, the effect of CGRP treatment on the viability of MC3T3-E1 cells were investigated. CGRP significantly stimulated the increase of viability of MC3T3-E1 cells in a dose-dependent manner with a maximal effect at 10 n M. Then MC3T3-E1 cells were pretreated with various concentrations of CGRP for 30 min, followed by treating with or without 500 ng/ml LPS for 48 h. The result showed that the cell viability inhibition by LPS could be significantly(P <0.01) reversed by CGRP with the concentration of 100 n M, but not 10 n M.(6). The cells were pretreated with or without 100 n M CGRP for 30 min prior to LPS-treatment or without LPS-treatment for 48 h. Flow cytometry analysis showed that, compared with non-treated culture, 500ng/ml LPS significantly(P<0.01) induced the apoptosis of MC3T3-E1 cells, which could be significantly(P<0.01) reversed by CGRP. Meanwhile, we also examined the effect of CGRP on the expression of cleaved caspase-3 protein in LPS-stimulated MC3T3-E1 cells.Western blot analysis also revealed that the enhanced level of cleaved caspase-3 by LPS was almost completely reduced by CGRP(P<0.01).(7).Previous studies has confirmed that, upon LPS stimulation, MAPK pathway including the p-JNK and the p- ERK were significantly promoted in MC3T3-E1 cells. As ERK pathway play a crucial role in a wide variety of cell functions especially cell proliferation and apoptosis, we investigated whether the anti-apoptotic effects of CGRP were related to inhibition of ERK signaling. The level of p-ERK in MC3T3-E1 cells induced by LPS was significantly downregulated by CGRP(P<0.01).(8).To further examine the role of ERK activation in the suppression of CGRP on LPS-induced apoptosis, both the inhibitor and agonist of ERK pathway were used. The western blotting results showed that treatment of MC3T3-E1 cells with ERK pathway-specific agonist C6-ceramide would lead to the reduction of inhibitory effect of CGRP on the LPS-induced p-ERK expression, while treatment with pathway-specific inhibitor PD98059 would result in a significant decrease in the p-ERK level(P<0.01). In parallel with these data, the flow cytometry analysis showed that, C6-ceramide could reduce the inhibitory effect of CGRP on LPS-induced apoptosis, and PD98059 could also reversed this change. Consistently, western blot analysis showed that, C6-ceramide would lead to the reduction of inhibitory effect of CGRP on LPS- triggered cleaved-caspase 3 level(P<0.01),while treatment with PD98059 would result in a significant decrease in the cleaved-caspase 3 level(P<0.01).(9).To investigate the potential protective effect of CGRP in periodontal tissue, an experimental periodontitis model of rat was established by LPS induction with or without CGRP intervention. After 4 weeks of LPS injection in periodontal tissue, micro-CT volumetric analysis was used to detect alveolar bone mass of the rat maxillae specimens. However, the LPS+CGRP group exhibited greater BVF value and lower bone resorption compared with those in the LPS group(P<0.01).Next, we focused on the inflammatory infiltration in the periodontal tissue. The tissue sections were stained with hematoxylin and eosin. Obvious inflammatory infiltration were observed in LPS group, while a relatively reduction was observed in LPS+CGRP group. Additionally, there were no obvious inflammatory infiltration in Control and CGRP groups.The Tissue sections were also subjected to IHC staining to detect the occurrence of apoptosis in the periodontal tissue.Conclusions:(1).CGRP promotes proliferation and differentiation in osteoblasts.(2).RAMP1 knockdown inhibited the CGRP-induced proliferation and osteoblastic differentiation in osteoblasts.(3).RAMP1 knockdown results in inhibition of differentiation via c AMP/PKA Pathway in osteoblasts.(4).CGRP significantly stimulated the increase of viability of MC3T3-E1 cells in a dose-dependent manner with a maximal effect at 10 n M.The cell viability inhibition by LPS could be significantly reversed by CGRP with the concentration of 100 n M, but not 10 n M.(5).CGRP attenuates the LPS-induced apoptosis in MC3T3-E1 cells.(6).CGRP attenuated LPS-induced p-ERK expression in MC3T3-E1 cells.(7).CGRP inhibited LPS-induced apoptosis and bone destruction in periodontitis model.