The Role Of H1-calponin In Mouse Bone Formation

There are two major forms of bone formation, intramembranous ossification and endochondral ossification. Both forms of ossification involve the condensation of mesenchyme, differentiation of mesenchymal cells into osteoblast, secretion and mineralization of extracellular matrix.h1-calponin(basic calponin)is one of the three distinct mammalian calponin genes. Calponins are closely related with the occurrence and development of multiple diseases. It has been discovered that h1-calponin may play an important role in skeleton development .Some researchers found that mice lacking h1-calponin showed increased bone formation in vivo and in vitro, which indicated that h1-calponin may be a negative regulator of bone formation, however the mechanism underlying the effects of h1-calponin on bone formation is still not being clarified.In this study, we first generated transgenic mice with overexpression of h1-calponin in osteoblasts(Mutant type, ColI-calponin ) to explore the role of h1-calponin in bone formation.MethodsPart I Generation of transgenic mice with overexpression of h1-calponin in osteoblasts1. The coding region of h1-calponin was amplified from mRNA of mouse kdney, then the cDNA of h1-calponin and the mouseα(1) I collagen promoter was cloned together into a SK plasmid containing SV40 polyA.The resulting vector, after being linearized using Sac II and Sal I, was injected into fertilized eggs to generate transgenic mice.2. Using PCR to screen mice with overexpression of h1-calponin in osteoblasts3.Osteoblast cells were isolated from the calvariae of wild-type and Col I–calponin mice. RT-PCRwas used to detect the expression of h1-calponin in cultured osteoblast cells. 4. The total RNA of several tissues was extracted and the expressions of h1-calponin were detected by RT-PCR to validate whether the overexpression of h1-calponin was osteoblast-specific.Part II The role of h1-calponin in bone formation1.Both wild type and mutant mice were observed for their body weight,body height and general health status.2. Dual beam X-Ray absorptiometry, Micro CT and three-point bending test were used to analyze the bone mass and biomechanical properties of femurs of adult mice.3. Histomorphometric measurement was used to evaluate osteoblastogenesis in vivo. Cell culture in vitro was used to detect the differentiation of osteoblasts. Reverse transcription PCR (RT-PCR) was used to measure the expression levels of several markers of osteogenesis.The level of Erk1/2 phosphorylation was detected by Western Blot. ResultsPart I Transgenic mouse strain (colI-calponin) with osteoblast-specifc overexpression of h1-calponin was generated successfully.1. h1-calponin cDNA was successfully inserted into genome of mutation mice, which ws confirmed by PCR detection of genomic DNA.2. The expression level of h1-calponin in osteoblasts of mutants, which was detected by using RT-PCR, was higher than that in WT mice.3. The specific overexpression of h1-calponin in osteoblasts were confirmed by detecting the expression of h1-calponin in multiples tissues of colI-calponin and WT mice.Part II Adult colI-calponin mice showed reduced bone mass and abnormal bone remodeling1. The adult colI-calponin mice showed lower bone mass and changed biomechanical properties of cortical bones.2. Proliferation and differentiation of osteoblasts in colI-calponin were decreased3. Mineralization of the cultured ostoblasts of ColI-calponin mice was decreased.4. The phosphorylation of Erk1/2 was enhanced in osteoblast of colI-calponin mie when compared with that inmutant mice.Conclusions:1. Transgenic mouse model with osteoblast-specific expression of h1-calponin was successfully constructed.2. ColI-calponin transgene mice had decreased bone mass, which may be caused by the dysregulated osteogenesis.3. Overexpression of h1-calponin in osteoblasts in vivo led to inhibited proliferation of osteoblasts, downregulateed expression level of OP and OC, and inhibited mineralization. The above-mentioned changes may be partially related to the enhanced activity of Erk1/2 signaling pathway.