右归丸对鼠膝骨性关节炎的治疗作用及Wnt信号通路相关因子表达的影响

目的：分析右归丸对膝骨性关节炎（KOA）大鼠Wnt信号通路相关因子表达的影响,探讨右归丸对KOA大鼠的保护机制。方法：SPF级SD大鼠60只,按照体重法随机分为假手术组、模型组、硫酸氨基葡萄糖组、右归丸高、中、低剂量组（n=10）。采用改良Hulth法复制膝骨关节炎大鼠模型。右归丸高中低剂量组分别按20、10、5 g/kg灌服相应的药物,硫酸氨基葡萄糖组按0.17 g/kg灌服硫酸氨基葡萄糖,假手术组和模型组灌服等体积的生理盐水,干预8周。末次给药后摘取膝关节,通过膝关节病理切片观察各组大鼠软骨组织病理改变;采用RT-PCR法对各组大鼠软骨组织DKK1、WISP1、Wnt1、β-catenin和LRP5 mRNA的表达水平进行对比分析;通过Western blot法检测各组大鼠软骨组织DKK1、WISP1、Wnt1、LRP5和β-catenin的蛋白质含量的变化。结果：与假手术组比较,模型组大鼠关节软骨受损严重,Mankin评分明显升高（P<0.05）;DKK1 mRNA表达水平和蛋白质表达水平明显降低（P<0.05）;WISP1、Wnt1、β-catenin、LRP5 mRNA表达水平及蛋白质表达水平明显升高（P<0.05）。与模型组比较,右归丸高剂量组和硫酸氨基葡萄糖组关节软骨病变明显减轻;Mankin评分明显减轻（P<0.05）;大鼠软骨组织中DKK1 mRNA表达水平和蛋白表达水平明显升高,WISP1、Wnt1、β-catenin、LRP5 mRNA及蛋白表达水平显著降低（P<0.05）。结论：右归丸通过抑制Wnt信号通路中WISP1、Wnt1、β-catenin、LRP5的表达,促进DKK1细胞因子的表达,发挥对KOA的保护作用。     

Essential role of beta-catenin in postnatal bone acquisition

Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in osteogenesis, we generated mice lacking the beta-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of beta-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of beta-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the beta-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-kappaB ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/beta-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.     

The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd

The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine Wnt.     

Beta-catenin levels influence rapid mechanical responses in osteoblasts

Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the molecular events involved in mechanical signal transduction are not well understood. Wnt/beta-catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce beta-catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on beta-catenin signaling. We found that mechanical strain caused a rapid, transient accumulation of active beta-catenin in the cytoplasm and its translocation to the nucleus. This was followed by up-regulation of the Wnt/beta-catenin target genes Wisp1 and Cox2, with peak responses at 4 and 1 h of strain, respectively. The increase of beta-catenin was temporally related to the activation of Akt and subsequent inactivation of GSK3beta, and caveolin-1 was not required for these molecular events. Application of Dkk-1, which disrupts canonical Wnt/LRP5 signaling, did not block strain-induced nuclear translocation of beta-catenin or up-regulation of Wisp1 and Cox2 expression. Conditions that increased basal beta-catenin levels, such as lithium chloride treatment or repression of caveolin-1 expression, were shown to enhance the effects of strain. In summary, mechanical strain activates Akt and inactivates GSK3beta to allow beta-catenin translocation, and Wnt signaling through LRP5 is not required for these strain-mediated responses. Thus, beta-catenin serves as both a modulator and effector of mechanical signals in bone cells.     

Gender specific LRP5 influences on trabecular bone structure and strength

A mutation in LRP5 (low-density lipoprotein receptor-related protein 5) has been shown to increase bone mass and density in humans and animals. Transgenic mice expressing the LRP5 mutation (G171V) demonstrate an increase in bone mass as compared to non-transgenic (NTG) littermates. This study evaluated LRP5 gene and gender-related influences on the structural and biomechanical strength properties of trabecular and cortical bone in femurs and vertebrae (L5) of 17-week-old mice. Micro-computed tomography was used to evaluate the trabecular bone structure of distal femurs and vertebrae ex vivo. Mechanical testing of the trabecular bone in the distal femur was done to determine biomechanical strength. Differences due to genotype and gender were tested using two-way ANOVA at a significance level of p<0.05. Trabecular bone structural parameters (BV/TV, trabecular thickness, number, etc.) at the distal femur, femoral neck, and vertebral body sites were greater in the transgenic as compared to the NTG mice. In addition, vertebral cortical thickness and trabecular strength parameters (ultimate and yield loads, stiffness, ultimate and yield stresses) in the distal femur were greater in the transgenic mice as compared to NTG. The increasing trends of cortical thickness were also noted in the transgenic mice as compared to NTG. Within LRP5 (G171V) mutant mice, there were significant gender-related differences in some of the trabecular bone structural parameters at all the sites (distal femur, femoral neck, and vertebral body). However, unlike trabecular structural parameters, the gender-specific differences were not found in the trabecular strength of LRP5 transgenic mice. In summary, these findings suggest that the LRP5 (G171V) mutation results in greater trabecular bone structure and strength at both the distal femurs and vertebral bodies as compared to NTG. In addition, only the trabecular structure parameters were affected by gender within the LRP5 (G171V) mutation.     

Wnt11/beta-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin

Current models of canonical Wnt signaling assume that a pathway is active if beta-catenin becomes nuclearly localized and Wnt target genes are transcribed. We show that, in Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. We provide evidence that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, we find that axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain beta-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes beta-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality.     

LDL receptor-related protein LRP6 regulates proliferation and survival through the Wnt cascade in vascular smooth muscle cells

Initial studies have established expression of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) in vascular smooth muscle cells (VSMCs). We hypothesized that LRP6 is a critical mediator governing the regulation of the canonical Wnt/beta-catenin/T cell factor 4 (Tcf-4) cascade in the vasculature. This hypothesis was based on our previous work demonstrating a role for the beta-catenin/Tcf-4 pathway in vascular remodeling as well as work in other cell systems establishing a role for LRP family members in the Wnt cascade. In line with our hypothesis, LRP6 upregulation significantly increased Wnt-1-induced Tcf activation. Moreover, a dominant interfering LRP6 mutant lacking the carboxyl intracellular domain (LRP6DeltaC) abolished Tcf activity. LRP6-induced stimulation of Tcf was blocked in VSMCs harboring constitutive expression of a dominant negative Tcf-4 transgene lacking the beta-catenin binding domain, suggesting that LRP6-induced activation of Tcf was mediated through a beta-catenin-dependent signal. Expression of the dominant interfering LRP6DeltaC transgene was sufficient to abolish the Wnt-induced survival as well as cyclin D1 activity and cell cycle progression. In conclusion, these findings provide the first evidence of a role for an LDL receptor-related protein in the regulation of VSMC proliferation and survival through the evolutionary conserved Wnt signaling cascade.     

Structure-based mutation analysis shows the importance of LRP5 beta-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling

A single point mutation (G to T) in the low-density lipoprotein receptor related protein 5 (LRP5) gene results in a glycine to valine amino acid change (G171V) and is responsible for an autosomal dominant high bone mass trait (HBM) in two independent kindreds. LRP5 acts as a co-receptor to Wnts with Frizzled family members and transduces Wnt-canonical signals which can be antagonized by LRP5 ligand, Dickkopf 1 (Dkk1). In the presence of Wnt1, LRP5 or the HBM variant (LRP5-G171V) induces beta-catenin nuclear translocation and activates T cell factor (TCF)-luciferase reporter activity. HBM variant suppresses Dkk1 function and this results in reduced inhibition of TCF activity as compared to that with LRP5. Structural analysis of LRP5 revealed that the HBM mutation lies in the 4th blade of the first beta-propeller domain. To elucidate the functional significance and consequence of the LRP5-G171V mutation in vitro, we took a structure-based approach to design 15 specific LRP5 point mutations. These included (a) substitutions at the G171 in blade 4, (b) mutations in blades 2-6 of beta-propeller 1, and (c) mutations in beta-propellers 2, 3 and 4. Here we show that substitutions of glycine at 171 to K, F, I and Q also resulted in HBM-like activity in the presence of Wnt1 and Dkk1. This indicates the importance of the G171 site rather than the effect of specific amino acid modification to LRP5 receptor function. Interestingly, G171 equivalent residue mutations in other blades of beta-propeller 1 (A65V, S127V, L200V, A214V and M282V) resulted in LRP5-G171V-like block of Dkk1 function. However G171V type mutations in other beta-propellers of LRP5 did not result in resistance to Dkk1 function. These results indicate the importance of LRP5 beta-propeller 1 for Dkk1 function and Wnt signaling. These data and additional comparative structural analysis of the LRP5 family member LDLR suggest a potential functional role of the first beta-propeller domain through intramolecular interaction with other domains of LRP5 wherein Dkk1 can bind. Such studies may also lead to a better understanding of the mechanisms underlying the reduced function of Dkk1-like inhibitory ligands of LRP5 with HBM-like mutations and its relationship to increased bone density phenotypes.     

Normal growth and development in mice over-expressing the CCN family member WISP3

Loss-of-function mutations in the gene WISP3 cause the autosomal recessive human skeletal disease Progressive Pseudorheumatoid Dysplasia, whereas mice with knockout mutations of Wisp3 have no phenotype. The lack of a phenotype in the Wisp3 knockout mice has constrained studies of the protein’s in vivo function. Over-expression experiments in zebrafish indicated that WISP3 may function as a BMP and Wnt signaling modulator. To determine whether these biologic activities are retained in mice, we created two strains of transgenic mice that over-express WISP3 in a broad array of tissues. Despite strong and persistent protein over-expression, the transgenic mice remained phenotypically indistinguishable from their non-transgenic littermates. Surprisingly, WISP3 contained in conditioned medium recovered from transgenic mouse primary kidney cell cultures was able to bind BMP and to inhibit BMP signaling in vitro. Factors that account for the difference between the in vitro and in vivo activities of WISP3 remain unknown. At present, the mouse remains a challenging model organism in which to explore the biologic function of WISP3.     

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.     

Up-regulation of the Wnt, estrogen receptor, insulin-like growth factor-I, and bone morphogenetic protein pathways in C57BL/6J osteoblasts as opposed to C3H/HeJ osteoblasts in part contributes to the differential anabolic response to fluid shear

C57BL/6J (B6), but not C3H/HeJ (C3H), mice responded to mechanical loading with an increase in bone formation. A 30-min steady fluid shear of 20 dynes/cm(2) increased [(3)H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin beta1 (Igtb1) and cyclooxygenase-2 (Cox-2)) in B6 but not C3H osteoblasts, indicating that the differential mechanosensitivity was intrinsic to osteoblasts. In-house microarray analysis with 5,500 gene fragments revealed that the expression of 669 genes in B6 osteoblasts and 474 genes in C3H osteoblasts was altered 4 h after the fluid shear. Several genes associated with the insulin-like growth factor (IGF)-I, the estrogen receptor (ER), the bone morphogenetic protein (BMP)/transforming growth factor-beta, and Wnt pathways were differentially up-regulated in B6 osteoblasts. In vitro mechanical loading also led to up-regulation of these genes in the bones of B6 but not C3H mice. Pretreatment of B6 osteoblasts with inhibitors of the Wnt pathway (endostatin), the BMP pathway (Noggin), or the ER pathway (ICI182780) blocked the fluid shear-induced proliferation. Inhibition of integrin and Cox-2 activation by echistatin and indomethacin, respectively, each blocked the fluid shear-induced up-regulation of genes associated with these four pathways. In summary, up-regulation of the IGF-I, ER, BMP, and Wnt pathways is involved in mechanotransduction. These four pathways are downstream to the early mechanoresponsive genes, i.e. Igtb1 and Cox-2. In conclusion, differential up-regulation of these anabolic pathways may in part contribute to the good and poor response, respectively, in the B6 and C3H mice to mechanical loading.