异补骨脂素促进小鼠胫骨骨折愈合的研究

目的:探讨灌服不同剂量异补骨脂素(isopsoralen,ISO)对小鼠骨折及血管愈合的影响。方法:选取2月龄体质量为(20±2)g的雄性C57BL/6小鼠60只,采用随机数字表法分为4组:模型组(Model)、低剂量组(isopsoralen-low dose,ISO-L)、中剂量组(isopsoralen-medium dose,ISO-M)和高剂量组(isopsoralen-high dose,ISO-H),每组15只。建立右侧胫骨骨折模型。术后采用灌胃给药,ISO-L组、ISO-M组和ISO-H组分别灌服ISO的浓度为10、20和40 mg·kg^-1,Model组灌服等体积生理盐水,每日1次,连续给药28 d。每周称量体重1次。分别于第7、14、21、28天行X线检查,并采用改良后I.R. Garrett评分对骨痂生长情况进行评价。至28 d后取材,剥离主要脏器称重并计算脏器系数,进行脏器苏木素伊红染色(hematoxylin-eosin,HE)观察病理结构变化。采用微计算机断层扫描技术(Micro-CT)扫描骨折区域并进行三维重建得出效果图,量化骨体积分数(bone volume/total volume,BV/TV)。将脱钙后的胫骨进行石蜡包埋并切片,通过HE染色和番红固绿染色观察骨折断端愈合及塑形情况。通过血管灌注Microfil造影剂后取出右侧胫骨并脱钙,使用Micro-CT扫描骨折区域骨痂微血管,比较血管体积分数及血管直径。结果:给药28 d后,各组小鼠体质量及器官系数比较,差异无统计学意义 (P＞0.05),脏器HE染色未发现明显病理学改变。X线片和改良后I.R. Garrett评分结果显示,ISO-M组28 d评分高于Model组(P＜0.05);ISO-H组第14、21、28 天评分均高于其他3组(P＜0.05)。Micro-CT结果显示,ISO-M组腔内骨痂明显减少,低于Model组(P＜0.05);ISO-H组骨痂大部分消退,BV/TV均低于其他3组(P＜0.05)。HE染色及番红固绿染色结果显示ISO-H组骨折区域断端闭合,已出现连续板层骨,骨折愈合进程超过其他组。血管造影结果显示,ISO-H组和ISO-M组血管体积分数高于Model组和ISO-L组(P＜0.05),ISO-H组和ISO-M组血管直径高于Model组和ISO-L组(P＜0.05)。结论:ISO在10~40 mg·kg^-1浓度范围内无明显毒副作用,可以改善骨微结构,促进骨痂微血管的生成,呈浓度依赖性加速小鼠骨折断端的愈合。     

杜仲多糖通过抑制NF-κB通路减轻IL-1β诱导的软骨细胞损伤

目的:探讨杜仲多糖对白细胞介素1β（interleukin-1β，IL-1β）诱导的软骨细胞损伤的影响及可能机制。方法：体外培养小鼠软骨细胞ATDC5，用含10μg/ml IL-1β处理ATDC5制作骨关节炎软骨细胞炎症模型，随机分为空白组、模型组、模型+杜仲多糖低浓度组、模型+杜仲多糖中浓度组和模型+杜仲多糖高浓度组。其中空白组细胞用常规培养基培养，模型组细胞用含10μg/ml IL-1β的培养基培养，模型+杜仲多糖低浓度组、模型+杜仲多糖中浓度组和模型+杜仲多糖高浓度组细胞分别用含100、200、400 μg/ml杜仲多糖与10μg/ml IL-1β的培养基共同培养。分别培养24、48、72 h后，CCK-8法检测细胞活力。培养48 h后，流式细胞术和DAPI染色检测细胞凋亡，ELISA法检测细胞培养上清液中肿瘤坏死因子α（tumor necrosis factor-α，TNF-α），一氧化氮（netric oxide，NO），γ干扰素（interfero-γ，IFN-γ）和白细胞介素6（interleukin-6，IL-6）的表达，DCFH-DA法检测细胞中活性氧含量，Western-blot法检测金属蛋白酶组织抑制因子1（tissue inhibrtor of metalloproteinase，TIMP-1），基质金属蛋白酶13（mitochondrial membrane protential，MMP-13）及NF-κB信号通路相关P65，磷酸化P65（p-P65）的蛋白表达，免疫荧光染色观察NF-κB P65细胞定位。结果：与空白组比较，模型组ATDC5细胞活力及TIMP-1蛋白表达降低（P<0.05），细胞凋亡率，TNF-α、NO、IFN-γ和IL-6水平，活性氧（reactive oxygen species，ROS）含量，MMP-13和p-P65的蛋白表达及细胞核内P65⁺数量均升高（P<0.05）。与模型组比较，模型+杜仲多糖低浓度组、模型+杜仲多糖中浓度组和模型+杜仲多糖高浓度组ATDC5细胞活力及TIMP-1蛋白表达升高（P<0.05），而细胞凋亡率、TNF-α、NO、IFN-γ和IL-6水平、ROS含量、MMP-13和p-P65的蛋白表达及细胞核内P65⁺数量均降低（P<0.05）。结论：杜仲多糖可促进白细胞介素1β诱导的软骨细胞ATDC5增殖，并抑制其凋亡、炎症反应和基质降解，其作用机制可能与抑制NF-κB通路的激活有关。     

RNA特异性腺苷脱氨酶1与Caveolin 1在脓毒症相关肝损伤中的作用及机制

背景与目的 脓毒症相关肝损伤（SRLI）发病机制尚不清楚,细菌内毒素（LPS）对肝脏血管内皮细胞的炎症损害可能是重要环节。前期研究提示,RNA特异性腺苷脱氨酶1（ADAR1）可能通过调控内皮细胞功能相关蛋白Caveolin 1（Cav-1）在参与血管内皮应激中的局部和全身炎症反应。因此,本研究初步探讨ADAR1与Cav-1在SRLI中的作用,以期为SRLI的早期防治寻找新的方法。方法 取ADARl基因敲除小鼠（ADAR1^ECKO）与野生型小鼠（ADAR1^flox/flox）各20只,腹腔注射LPS（20 mg/kg）诱导脓毒症小鼠模型脓毒症模型,6 h后每组小鼠各取10只,获取肝脏组织,并分离肝窦内皮细胞（LSECs）,通过HE染色观察其肝脏病理学改变,用细胞免疫荧光法观察LSECs中Cav-1及其下游蛋白VE-cadherin的表达,两组其余小鼠用于生存率分析;用ADARl siRNA转染正常野生型小鼠的LSECs后,通过内皮细胞成管实验观察转染后LSECs的增殖情况、Western blot检测Cav-1下游相关蛋白的表达。结果 生存观察结果显示,注射LPS后,ADAR1^ECKO小鼠死亡时间早于ADAR1^flox/flox小鼠,存活率低于ADAR1^flox/flox小鼠（均P<0.05）;组织病理学观察显示,注射LPS 6 h后,ADAR1^ECKO小鼠的肝损伤比ADAR1^flox/flox小鼠更严重;细胞免疫荧光观察显示,注射LPS 6 h后,ADAR1^ECKO小鼠LSECs中Cav-1与VE-cadherin的表达低于ADAR1^flox/flox小鼠。正常野生型小鼠的LSECs转染ADARl siRNA后,成管能力明显减弱,Cav-1下游蛋白VE-cadherin的表达下调,但β-Catenin的表达无明显变化。结论 ADAR1的下调或功能缺失会导致SRLI加重,机制可能涉及其调控Cav-1/VE-cadherin通路的活性。因此,激活ADAR1/Cav-1/VE-cadherin通路可能是防治SRLI的有效策略。     

软骨细胞条件性敲除PTEN基因的FGFR3功能增强型点突变小鼠的获得及其表型初步分析

目的 获得在软骨细胞中条件性敲除PIEN基因的FGFR3增强型点突变小鼠并进行初步表型分析.方法 通过FTEN条件性基因敲除小鼠(Ptenflox/flox小鼠),与软骨细胞特异表达Cre重组酶的小鼠(Col2aCre)交配,获得在软骨细胞中特异敲除PTEN基因小鼠(Col2aCre:Ptenfolox/flox);同时,利用Ptenflox/flox小鼠与FGFR3增强型点突变小鼠(Fgfr3G369C/+小鼠,即ACH小鼠)交配,子代杂合子小鼠(Ptenflax/:ACH)之间再交配,获得Ptenflox/flox:ACH小鼠;通过Col2aCre:Ptenflox/flox和Ptenflox/flox:ACH交配即可获得在软骨细胞中特异性敲除PTEN基因的FGFB3增强型点突变小鼠((Col2aCre:Ptenflox/flox:ACH).采用PCR对小鼠基冈型进行鉴定,免疫荧光染色检测PTEN基因的敲除效率,通过X光平片摄影对小鼠的表型进行初步分析.结果 获得了在软骨细胞中敲除PTEN的FGFR3点突变小鼠,免疫荧光染色证实Col2aCre:Ptenflox/flox:ACH小鼠软骨细胞中PTEN蛋白因为基因敲除而表达很低.初步的表型分析显示:Col2aCre:Ptenflox/flox:ACH小鼠身长、尾长均较Pten flox/flox:ACH小鼠长(P＜0.05,n=5).Col2aCre:Ptenflox/flox:ACH小鼠表型,较Ptenflox/flox:ACH小鼠的侏儒表型有所缓解.结论 采用基于Cre/LoxP系统的条件性基因敲除策略,获得了在软骨细胞中敲除PTEN基因的FGFR3增强型突变小鼠,表型分析初步显示,软骨细胞特异性敲除PTEN基因可部分缓解由FGFR3增强型点突变所导致的小鼠侏儒表型.该小鼠的获得,为研究P13K/AKT信号通路在FGFR3突变介导的软骨生长抑制中的作用提供了实验动物平台.     

多靶点沉默SOST基因修饰MC3T3-E1细胞注射对小鼠椎体微结构的影响

目的 利用多靶点SOST基因沉默技术修饰小鼠胚胎成骨细胞前体细胞MC3T3-E1,并将其注射到小鼠体内,观察其对小鼠椎体骨密度及微结构的影响。方法 取18只18月龄雌性C57BL/6小鼠在无菌条件下手术切除双侧卵巢制作骨质疏松模型,并分为3组（n=6）：SOST基因沉默组注射采用多靶点SOST基因沉默技术修饰并同时转染CRISPR-sgRNA载体系统和SOST-RNAi修复模板载体假病毒颗粒的MC3T3-E1细胞;阴性组注射同时转染CRISPR-sgRNA载体系统和SOST-N修复模板载体假病毒颗粒的MC3T3-E1细胞;空白组注射不作任何处理的MC3T3-E1细胞。采用HE染色和Masson三色染色观察小鼠椎体骨小梁面积的变化,免疫组化染色观察骨组织中骨保护素（OPG）和RANKL表达的变化,实时荧光定量PCR检测观察各组细胞SOST及骨代谢相关细胞因子（RUNX2、β-catenin、RANKL、OPG）的表达变化。结果 L₁ HE染色和L₂ Masson三色染色结果显示,SOST基因沉默组骨小梁面积大于阴性组和空白组,差异均有统计学意义（P < 0.05）。L₂免疫组织化学染色结果显示,SOST基因沉默组OPG表达量高于阴性组和空白组,RANKL表达量低于阴性组和空白组,差异均有统计学意义（P < 0.05）。L₃实时荧光定量PCR检测结果显示,SOST基因沉默组SOST和RANKL表达量低于阴性组,RUNX2、β-catenin和OPG表达量高于阴性组,差异均有统计学意义（P < 0.05）。结论 多靶点沉默SOST基因修饰MC3T3-E1细胞尾静脉注射能够促进小鼠成骨细胞分化和分泌功能,抑制破骨细胞活性,有效改善小鼠椎体微结构。     

Cell cycle regulation of human pancreatic cancer by tamoxifen

Background: Clinical trials have suggested a survival advantage for selected patients with metastatic pancreatic cancer treated with tamoxifen. We sought to identify the molecular mechanism by which tamoxifen inhibits human pancreatic cancer cell (HPCC) growth. Methods: HPCCs were grown in tamoxifen and growth inhibition was determined by³H-thymidine uptake and by the MTT assay; changes in cell viability were determined by cell counts. Cell cycle alterations were evaluated by FACS, and the induction of apoptosis was evaluated using the TUNEL assay. Total cellular RNA was isolated after tamoxifen treatment, and Northern blot analysis was performed for p21 ^waf1 . Results: Tamoxifen inhibited HPCC growth as measured by inhibition of³H-thymidine incorporation and by the MTT assay. However, there was no decrease in the total number of viable cells after 6 days of treatment with 10 μM of tamoxifen and no evident apoptosis, confirming the absence of a cytotoxic effect. Cell cycle analysis revealed cellular arrest in the G₀/G₁ phase, which correlated with p21 ^waf1 mRNA upregulation in response to tamoxifen treatment. Conclusions: Tamoxifen inhibits HPCC growth by inducing G₀/G₁ arrest with an associated increase in p21 ^waf1 mRNA expression. Tamoxifen is an effective inhibitor of HPCC growth in vitro and warrants further in vivo study.     

125I粒子抑制裸鼠T24移形细胞癌效果

目的 观察不同活度¹²⁵I粒子抑制裸鼠T24移形细胞癌的效果。方法 将40只接种T24人移形细胞癌株荷瘤裸鼠均分为高、中、低活度组和对照组,每组10只,分别于肿瘤中心植入1枚活度0.9 mCi（33.3 MBq）、0.6 mCi（22.2 MBq）、0.3 mCi（11.1 MBq）和0 mCi（粒子不含核素）的¹²⁵I粒子。检测并对比各组植入10、20天后90%肿瘤组织吸收剂量（D₉₀）、抑瘤率（IR）、HE染色放射治疗反应分级（RRG）、凋亡指数及B淋巴细胞瘤-2（Bcl-2）蛋白表达。结果 ¹²⁵I粒子植入后10、20天,各组D₉₀及IR均逐渐降低（P均<0.05）,¹²⁵I粒子周围5 mm以内肿瘤组织均见明显坏死,粒子活度越高、时间越长,坏死范围越大;同期各组凋亡指数均逐渐降低,Bcl-2蛋白表达逐渐增加（P均<0.05）。结论 ¹²⁵I粒子能明显抑制裸鼠T24移形细胞癌生长,促进肿瘤细胞凋亡是其作用机制之一。     

原发性中枢神经系统淋巴瘤Toll样受体4表达水平与弥散加权成像表观弥散系数值的相关性

目的 观察原发性中枢神经系统淋巴瘤(PCNSL)Toll样受体4(TLR4)表达水平与MR弥散加权成像(DWI)表观弥散系数(ADC)值的相关性。方法 回顾性分析17例经病理确诊的PCNSL患者,观察病灶ADC值、TLR4及NF-κB表达水平之间的相关性。结果 PCNSL病灶ADC值为460×10^－6~1 034×10^－6 mm²/s,平均(756±147)×10^－6 mm²/s;TLR4表达水平为0.02~0.11,平均0.06±0.03,NF-κB表达水平为0.04~0.15,平均0.08±0.03。TLR4表达水平与ADC值呈负相关(r=－0.76,P<0.01);NF-κB表达水平与TLR4及ADC值之间无明显相关性(P均>0.05)。结论 PCNSL的TLR4表达水平与其ADC值呈负相关。     

基于开源软件Horos的颈椎椎动脉V2段解剖变异研究

目的 分析颈椎椎动脉V2段解剖变异情况。方法 利用个人电脑在Horos软件中对169例中国成人头颈部CT血管造影（CT angiography, CTA）影像学资料进行分析,其中男119例,女50例。将椎动脉V2段首先穿行的横突孔并非C6者定义为走行变异,分析其发生率的侧别差异和性别差异;将椎动脉内壁距中线距离不足1 cm者定义为椎动脉V2段内聚,分析其发生率的侧别差异和性别差异。结果 169例病人的双侧椎动脉（共338侧）中,32侧存在走行变异,其中椎动脉缺如者4侧;椎动脉首先穿行C₄、C₅横突孔者分别为5侧、23侧,4例病人双侧椎动脉同时存在走行变异,均首先穿行C₅横突孔;走行变异存在于左侧者为16侧,存在于右侧者为16侧,差异无统计学意义（P＞0.05）;男性病人椎动脉走行变异发生率为16.81%（20/119）,女性病人椎动脉走行变异发生率为16.00%（8/50）,差异无统计学意义（χ²=0.017,P=0.898）。共11例病人的椎动脉V2段存在内聚,均为单侧,其中左侧8例（8/169,4.73%）,右侧3例（3/169,1.78%）,侧别分布差异无统计学意义（χ²=1.503,P=0.221）;11例病人中男性为3例（3/119,2.52%）,女性为8例（8/50,16.00%）,性别分布差异有统计学意义（χ²=8.413,P=0.004）。结论 椎动脉V2段的解剖结构和走行路径存在变异,首先穿行的横突孔可能位于C₅或C₄,走行变异的发生率无侧别或性别差异;V2段可发生内聚,使其更靠近中线,其发生率无侧别差异,但有性别差异,女性病人多发。以上变异可能给颈椎手术或穿刺等微创手术相关的操作带来风险,应在术前通过辅助检查明确。     

TAK1 regulates cartilage and joint development via the MAPK and BMP signaling pathways

The importance of canonical transforming growth factor β (TGF‐β) and bone morphogenetic protein (BMP) signaling during cartilage and joint development is well established, but the necessity for noncanonical (SMAD‐independent) signaling during these processes is largely unknown. TGF‐β activated kinase 1 (TAK1) is a MAP3K activated by TGF‐β, BMP, and other mitogen‐activated protein kinase (MAPK) signaling components. We set out to define the potential role for noncanonical, TAK1‐mediated signaling in cartilage and joint development via deletion of Tak1 in chondrocytes (Col2Cre;Tak1^f/f) and the developing limb mesenchyme (Prx1Cre;Tak1^f/f). Deletion of Tak1 in chondrocytes resulted in novel embryonic developmental cartilage defects including decreased chondrocyte proliferation, reduced proliferating chondrocyte survival, delayed onset of hypertrophy, reduced Mmp13 expression, and a failure to maintain interzone cells of the elbow joint, which were not observed previously in another Col2Cre;Tak1^f/f model. Deletion of Tak1 in limb mesenchyme resulted in widespread joint fusions likely owing to the differentiation of interzone cells to the chondrocyte lineage. The Prx1Cre;Tak1^f/f model also allowed us to identify novel columnar chondrocyte organization and terminal maturation defects owing to the interplay between chondrocytes and the surrounding mesenchyme. Furthermore, both our in vivo models and in vitro cell culture studies demonstrate that loss of Tak1 results in impaired activation of the downstream MAPK target p38, as well as diminished activation of the BMP/SMAD signaling pathway. Taken together, these data demonstrate that TAK1 is a critical regulator of both MAPK and BMP signaling and is necessary for proper cartilage and joint development. © 2010 American Society for Bone and Mineral Research     

Conditional deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation,increasing volume of remodeling bone in mice

Bone undergoes remodeling consisting of osteoclastic bone resorption followed by osteoblastic bone formation throughout life. Although the effects of bone morphogenetic protein (BMP) signals on osteoblasts have been studied extensively, the function of BMP signals in osteoclasts has not been fully elucidated. To delineate the function of BMP signals in osteoclasts during bone remodeling, we deleted BMP receptor type IA (Bmpr1a) in an osteoclast‐specific manner using a knock‐in Cre mouse line to the cathepsin K locus (Ctsk^Cre/+;Bmpr1a^flox/flox, designated as Bmpr1a^ΔOc/ΔOc). Cre was specifically expressed in multinucleated osteoclasts in vivo. Cre‐dependent deletion of the Bmpr1a gene occurred at 4 days after cultivation of bone marrow macrophages obtained from Bmpr1a^ΔOc/ΔOc with RANKL. These results suggested that Bmpr1a was deleted after formation of osteoclasts in Bmpr1a^ΔOc/ΔOc mice. Expression of bone‐resorption markers increased, thus suggesting that BMPRIA signaling negatively regulates osteoclast differentiation. Trabeculae in tibia and femurs were thickened in 3.5‐, 8‐, and 12‐week‐old Bmpr1a^ΔOc/ΔOc mice. Bone histomorphometry revealed increased bone volume associated with increased osteoblastic bone‐formation rates (BFR) in the remodeling bone of the secondary spongiosa in Bmpr1a^ΔOc/ΔOc tibias at 8 weeks of age. For comparison, we also induced an osteoblast‐specific deletion of Bmpr1a using Col1a1‐Cre. The resulting mice showed increased bone volume with marked decreases in BFR in tibias at 8 weeks of age. These results indicate that deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation, thus suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone‐formation activity during bone remodeling. © 2011 American Society for Bone and Mineral Research     

Epidermal growth factor receptor plays an anabolic role in bone metabolism in vivo

While the epidermal growth factor receptor (EGFR)–mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro–computed tomography (µCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col‐Cre Egfr^f/f), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant‐negative allele, Wa5, and generated Col‐Cre Egfr^Wa5/f mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony‐forming unit–fibroblast (CFU‐F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild‐type mice caused a significant reduction in trabecular bone volume. In contrast, Egfr^Dsk5/+ mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism. © 2011 American Society for Bone and Mineral Research.     

Loss of Sprouty Produces a Ciliopathic Skeletal Phenotype in Mice Through Upregulation of Hedgehog Signaling

The Sprouty family is a highly conserved group of intracellular modulators of receptor tyrosine kinase (RTK)-signaling pathways, which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders called ciliopathies. We aimed to evaluate Sprouty2 and Sprouty4 gene-dependent alterations of ciliary structure and to focus on the determination of its association with Hedgehog signaling defects in chondrocytes. Analysis of the transgenic mice phenotype with Sprouty2 and Sprouty4 deficiency revealed several defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, reduced bone thickness and trabecular bone mass, skull deformities, or chondroma-like lesions were revealed. All these pathologies might be attributed to ciliopathies. Elongation of the ciliary axonemes in embryonic and postnatal growth plate chondrocytes was observed in Sprouty2^−/− and Sprouty2^+/−/Sprouty4^−/− mutants compared with corresponding littermate controls. Also, cilia-dependent Hedgehog signaling was upregulated in Sprouty2/4 mutant animals. Ptch1 and Ihh expression were upregulated in the autopodium and the proximal tibia of Sprouty2^−/−/Sprouty4^−/− mutants. Increased levels of the GLI3 repressor (GLI3R) form were detected in Sprouty2/4 mutant primary fibroblast embryonic cell cultures and tissues. These findings demonstrate that mouse lines deficient in Sprouty proteins manifest phenotypic features resembling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of RTK and dysfunction of primary cilia during skeletogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Cbfb Regulates Bone Development by Stabilizing Runx Family Proteins

Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA‐binding capacity. Cbfb‐deficient (Cbfb^?/?) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb^?/? mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb^fl/fl mice with Dermo1 Cre knock‐in mice. Cbfb^fl/fl/Cre mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb^fl/fl/Cre chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb^fl/fl/Cre embryos compared with the respective protein in the respective tissue of Cbfb^fl/fl embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb^fl/fl/Cre primary osteoblasts, and Runx2 protein was less stable in Cbfb^fl/fl/Cre osteoblasts than Cbfb^fl/fl osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons. © 2014 American Society for Bone and Mineral Research.     

Activation of β‐Catenin Signaling in Articular Chondrocytes Leads to Osteoarthritis‐Like Phenotype in Adult β‐Catenin Conditional Activation Mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/β‐catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β‐catenin in this disease has not been reported. Because tissue‐specific activation of the β‐catenin gene (targeted by Col2a1‐Cre) is embryonic lethal, we specifically activated the β‐catenin gene in articular chondrocytes in adult mice by generating β‐catenin conditional activation (cAct) mice through breeding of β‐catenin^{fx(Ex3)/fx(Ex3)} mice with Col2a1‐CreER^T2 transgenic mice. Deletion of exon 3 of the β‐catenin gene results in the production of a stabilized fusion β‐catenin protein that is resistant to phosphorylation by GSK‐3β. In this study, tamoxifen was administered to the 3‐ and 6‐mo‐old Col2a1‐CreER^T2;β‐catenin^fx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β‐catenin protein was detected by immunostaining in articular cartilage tissues of β‐catenin cAct mice. In 5‐mo‐old β‐catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8‐mo‐old β‐catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in β‐catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp‐9, Mmp‐13, Alp, Oc, and colX, was significantly increased (3‐ to 6‐fold) in articular chondrocytes derived from β‐catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6‐fold increase) in these cells. In addition, we also observed overexpression of β‐catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β‐catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA‐like phenotype. This study provides direct and definitive evidence about the role of β‐catenin in the development of OA.