■含锌指的转录因子Osterix/特异性蛋白-7(Sp7)是成骨细胞分化的必需转录因子。然而,其在分化成骨细胞中的功能尚不清楚,成骨细胞特异性Sp7缺失对骨细胞的影响尚未得到充分研究.
■Sp7floxneo/floxneo小鼠,其中Sp7表达是野生型小鼠的30%,因为neo基因插入干扰了剪接,和使用2.3kbCol1a1增强绿色荧光蛋白(EGFP)-Cre的成骨细胞特异性敲除(Sp7fl/fl;Col1a1-Cre)小鼠通过显微计算机断层扫描(micro-CT)检查,骨组织形态计量学,血清标记物,和组织学分析。通过实时逆转录(RT)-PCR分析检测成骨细胞和骨细胞标记基因的表达。成骨,破骨细胞生成,并检查了原代成骨细胞中I型胶原α1链(Col1a1)的表达调节。
■雌性Sp7floxneo/floxneo和Sp7fl/fl;Col1a1-Cre小鼠的股骨小梁骨体积高于相应的对照组,但不是男性。雄性Sp7fl/fl中溴脱氧尿苷(BrdU)阳性成骨细胞增加;Col1a1-Cre小鼠,雌性Sp7fl/fl的胫骨骨小梁中成骨细胞数量和骨形成率增加;Col1a1-Cre小鼠,尽管成骨细胞成熟在雌性Sp7fl/fl中受到抑制;Col1a1-Cre小鼠,如未成熟成骨细胞标记基因的表达增加所示,分泌磷蛋白1(Spp1),成熟成骨细胞标记基因的表达降低,骨γ-羧基谷氨酸蛋白/骨γ-羧基谷氨酸蛋白2(Bglap/Bglap2)。此外,在Sp7fl/fl的原代成骨细胞培养物中,碱性磷酸酶活性增加,但矿化减少;Col1a1-Cre小鼠。因此,Sp7fl/fl中积累的未成熟成骨细胞;Col1a1-Cre小鼠可能补偿了雄性和雌性不同水平的成骨细胞成熟抑制。雄性和雌性Sp7fl/fl的椎体骨小梁体积均较低;Col1a1-Cre小鼠比对照组低,Sp7fl/fl中雌性成骨细胞参数和骨形成率较低;Col1a1-Cre小鼠比Sp7fl/fl小鼠低,提示长骨和椎骨的不同调节机制。Sp7floxneo/floxneo和Sp7fl/fl中的股骨皮质骨薄而多孔;两种性别的Col1a1-Cre小鼠,小管的数量减少了,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)阳性腔隙和破骨细胞增加,而Sp7fl/fl的骨形成速率相似;Col1a1-Cre和Sp7fl/fl小鼠。血清总1型前胶原N端前肽(P1NP)水平,骨形成的标记,相似,而那些抗酒石酸酸性磷酸酶5b(TRAP5b),骨吸收的标记,在Sp7fl/fl中更高;Col1a1-Cre小鼠。成骨细胞较少长方体,Col1a1和Col1a1-EGFP-Cre在Sp7fl/fl中的表达较低;Col1a1-Cre小鼠,Sp7过表达诱导Col1a1表达。
■我们的研究表明,Sp7抑制未成熟成骨细胞的增殖,诱导成骨细胞成熟和Col1a1表达,并且是骨细胞获得足够数量的存活过程所必需的,防止皮质孔隙。
■本研究阐明了Sp7在增殖分化成骨细胞中的作用,成熟,Col1a1表达式,和骨细胞形成过程,在开发骨质疏松症的治疗方法中需要靶向SP7。
UNASSIGNED: Zinc finger-containing transcription factor Osterix/Specificity protein-7 (Sp7) is an essential transcription factor for osteoblast differentiation. However, its functions in differentiated osteoblasts remain unclear and the effects of osteoblast-specific Sp7 deletion on osteocytes have not been sufficiently studied.
UNASSIGNED: Sp7 floxneo/floxneo mice, in which
Sp7 expression was 30 % of that in wild-type mice because of disturbed splicing by neo gene insertion, and osteoblast-specific knockout (Sp7 fl/fl;Col1a1-Cre) mice using 2.3-kb Col1a1 enhanced green fluorescent protein (EGFP)-Cre were examined by micro-computed tomography (micro-CT), bone histomorphometry, serum markers, and histological analyses. The expression of osteoblast and osteocyte marker genes was examined by real-time reverse transcription (RT)-PCR analysis. Osteoblastogenesis, osteoclastogenesis, and regulation of the expression of collagen type I alpha 1 chain (Col1a1) were examined in primary osteoblasts.
UNASSIGNED: Femoral trabecular bone volume was higher in female
Sp7 floxneo/floxneo and
Sp7 fl/fl;Col1a1-Cre mice than in the respective controls, but not in males. Bromodeoxyuridine (BrdU)-positive osteoblastic cells were increased in male Sp7 fl/fl;Col1a1-Cre mice, and osteoblast number and the bone formation rate were increased in tibial trabecular bone in female Sp7 fl/fl;Col1a1-Cre mice, although osteoblast maturation was inhibited in female Sp7 fl/fl;Col1a1-Cre mice as shown by the increased expression of an immature osteoblast marker gene, secreted phosphoprotein 1 (Spp1), and reduced expression of a mature osteoblast marker gene, bone gamma-carboxyglutamate protein/bone gamma-carboxyglutamate protein 2 (Bglap/Bglap2). Furthermore, alkaline phosphatase activity was increased but mineralization was reduced in the culture of primary osteoblasts from Sp7 fl/fl;Col1a1-Cre mice. Therefore, the accumulated immature osteoblasts in Sp7 fl/fl;Col1a1-Cre mice was likely compensated for the inhibition of osteoblast maturation at different levels in males and females. Vertebral trabecular bone volume was lower in both male and female Sp7 fl/fl;Col1a1-Cre mice than in the controls and the osteoblast parameters and bone formation rate in females were lower in Sp7 fl/fl;Col1a1-Cre mice than in Sp7 fl/fl mice, suggesting differential regulatory mechanisms in long bones and vertebrae. The femoral cortical bone was thin and porous in
Sp7 floxneo/floxneo and
Sp7 fl/fl;Col1a1-Cre mice of both sexes, the number of canaliculi was reduced, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive lacunae and the osteoclasts were increased, whereas the bone formation rate was similar in Sp7 fl/fl;Col1a1-Cre and
Sp7 fl/fl mice. The serum levels of total procollagen type 1 N-terminal propeptide (P1NP), a marker for bone formation, were similar, while those of tartrate-resistant acid phosphatase 5b (TRAP5b), a marker for bone resorption, were higher in Sp7 fl/fl;Col1a1-Cre mice. Osteoblasts were less cuboidal, the expression of Col1a1 and Col1a1-EGFP-Cre was lower in Sp7 fl/fl;Col1a1-Cre mice, and overexpression of
Sp7 induced Col1a1 expression.
UNASSIGNED: Our studies indicated that Sp7 inhibits the proliferation of immature osteoblasts, induces osteoblast maturation and Col1a1 expression, and is required for osteocytes to acquire a sufficient number of processes for their survival, which prevents cortical porosity.
UNASSIGNED: This study clarified the roles of Sp7 in differentiated osteoblasts in proliferarion, maturation, Col1a1 expression, and osteocyte process formation, which are required for targeting SP7 in the development of therapies for osteoporosis.