Role Of Cathepsin K In The Formation Of Dental Hard Tissues

Cathepsin K(CTSK),also called lysosomal cysteine cathepsin K,is a member of the papain like cysteine protease family first founded in rabbit's osteoclasts.At first,CTSK is considered to be mainly expressed in osteoclasts,and through the degradation of type I collagen,osteopontin and bone matrix proteins in the acidic environment to play an important role in osteoclast-mediated bone resorption.In clinical genetic studies,CTSK gene is located in the first chromosome,and its mutations have been found to cause pycnodysostosis,an autosomal recessive bone disease.The typical features of pycnodysostosis include increased bone density,short stature,osteolysis of the distal phalanges,frequent pathologic fractures,as well as dental abnormalities,such as hypoplasia of enamel,hypercementosis,obliterated pulp chambers and periodontal disease.But its mechanism is not clear,and even the expression pattern and function of CTSK in odontogenesis has not been reported yet.Considering that the bone and dental hard tissues(enamel,dentin and cementum)have similar composition and physicochemical properties,CTSK may also play an important role in the formation of dental hard tissue.In this study,the expression pattern of CTSK in the process of odontogenesis was taken as the breakthrough point,to find the possible zymolyte of CTSK in odontogenesis in vitro degradation test.And we built the Ctsk-knockout mice via CRISPR/Cas9 and analyzed their phenotype,and through the research on CTSK gene mutation of clinical cases,to explore the role of CTSK in the formation of dental hard tissues,which lays the foundation for the further research of the odontogenesis mechanism.Methods: 1)A total of twenty-five BALB/c mice,divided into five groups: the embryonic day 18(E18),post-natal day 1(P1),P5,P10 and P20(5 mice at each group).After intraperitoneal injection with 1% Pentobarbital Sodium,mandibles were isolated from the narcotized mice.Tissue preparation,paraffin section,deparaffinization and rehydration were performed according to previously described methods.Antigen retrieval,blocking of the activity of endogenous tissue peroxidase,non-specific binding,primary antibody incubation,and immunohistochemistry staining were performed using Read-to-Use SABC-POD.Immunoperoxidase staining was performed according to the manufacture's protocol.2)The zymography was performed using gels with 0.1%(w/v)Emdogain.In brief,different amount(0.015 ?g,0.045 ?g,0.075 ?g,0.105 ?g,0.15 ?g,0.225 ?g respectively)of Active human Procathepsin K protein fragment was mixed with sterilized deionized water to 10 ?L and then mixed with 2.5 ?L of 5 × SDS-sample buffer.Then electrophoresis was conducted with a Bio-Rad Mini-Protein system with a constant voltage of 120 V at 4 °C.After electrophoresis,in order to remove SDS,the gels were immersed in 2.5%(v/v)Triton X-100 for 1 hrs,washed twice in the three groups of incubation buffer(50 m M sodium acetate,2.5 m M dithiothreitol(DTT),2.5 m M EDTA)(p H=4.5,p H=5.5 p H=8 respectively),and then immersed in the incubation buffer at 37 °C for 2,6,12,24,48 hrs,respectively.The gels were subsequently washed with water and stained in 45% methanol /10% acetic acid/water containing 0.25% Coomassie Brilliant Blue R-250.3)Two micrograms(0.25 ?g/?L)of Human Amelogenin,X-Linked(AMELX)full length protein with 0.15 ?g or 0.025 ?g of Active human Procathepsin K protein fragment(0.15 ?g/?L)was incubated in 20 ?L reactions in incubation buffer as mentioned above for 0,2,4,8,12,24,36 and 48 hrs at 37 ?C.The reaction was ended by boiling for 5 min mixed with 5 ?L of 5× SDS-sample buffer with reducing agents and analyzed by SDS–PAGE.Following electrophoresis,silver staining was performed according to the literature Handbook of Protein Technology.4)Commissioned by Shanghai Biomodel Organism Science & Technology Development Co.,Ltd.,built the Ctsk-knockout mice via CRISPR/Cas9 system.5)The morphology and structure of mandible and teeth of CTSK-knockout mice were observed through the Micro CT and stereomicroscope.6)Through the history collection,physical examination,pathological examination,X-ray examination,CBCT and gene sequencing,we finish a clinical research and gene analysis in a patient with pycnodysostosis.Results: 1)This study showed that CTSK was first present in trace amounts in pre-ameloblasts during the pre-secretory stage.After that,a peak expression was then detected in secretory ameloblasts,and continued to be expressed until the late maturation stage.2)Primary odontoblasts had very low level of CTSK during the early stage of dentin formation.Then a strong immunostaining was detected in secretory odontoblasts during the dentin mineralization stage,and persisted to the late stage of dentinogenesis.However,CTSK was not found in the odontoblasts after tooth eruption.3)CTSK was proved to be capable of hydrolyzing 0.1% Emdogain.As time going by,the proteolysis regions became clearer and clearer.The enzymatic hydrolysis ability increased with the increase of CTSK concentration and reaction time.4)CTSK was able to split AMELX into multiple cleavage products in both molars with a ratio of 1:13(0.15 ?g: 2 ?g)and 1:80(0.025 ?g: 2 ?g).The enzymatic hydrolysis ability increased with the increase of CTSK concentration and reaction time.5)Building the Ctsk-knockout mice to provide an experimental tool for studying the role of CTSK in the odontogenesis mechanism.6)We observed that the molar of Ctsk-/-mice showed dental hard tissue abnormalities,such as the hypomaturation of enamel hypoplasia,and dentin thickening led to obliterated pulp chambers.7)Patients diagnosed pycnodysostosis displayed multiple abnormalities,such as short stature,increased bone density,collarbone dysplasia,short hands and feet,dysplastic nails,finger / toe bone resorption,frontal swelling,facial zygomatic collapse,mandivble hypoplasia,obtuse mandibular angle,palatal vault disappeared,maxillary dentition defect,mandibular dentition loss,abnormality of tooth eruption,hypercementosis,mandibular osteomyelitis.Identified heterozygous mutation site(p.Gly146 Asp and p.Tyr203His).Conclusion: The present study mainly finds CTSK is present in ameloblasts and odontoblasts,and finds the expression pattern of CTSK in tooth germ and its function in degrading enamel matrix proteins.We built the Ctsk-knockout mice successfully to provide an experimental tool for studying the role of CTSK in the odontogenesis mechanism.This is the first time we observed that the molar of Ctsk-/-mice showed dental hard tissue abnormalities,such as the hypomaturation of enamel hypoplasia,and dentin thickening led to obliterated pulp chambers.And we also confirm that the pycnodysostosis patient caused by CTSK mutation exhibited a variety of symptoms,such as abnormal odontogenesis.In conclusion,we proved CTSK plays a role in the formation of dental hard tissues,lay the foundation for the further study on the pathogenesis of the dental hard tissue abnormalities in the pycnodysostosis and to reveal the mechanism of CTSK in the formation of dental hard tissue.