Function And Mechanism Of PDZK1 Regulating Osteoclast And Osteoblast Differentiation

Part Ⅰ: The effect of PDZK1 on ovariectomized mice model【Objects】Postmenopausal osteoporosis(PMOP)is a disorder of bone metabolism caused by a decrease in the level of estrogen,which has a protective effect on bone.When estrogen levels decrease,osteoclast activity increases and osteogenesis is insufficient,resulting in increased bone conversion rate,decreased bone density and bone microstructure changes,resulting in increased bone fragility,easy fracture,seriously affecting the health and living standards of patients.The results of epidemiological investigation showed that the incidence of PMOP increased year by year.However,the therapeutic means of PMOP are still relatively limited.Further understanding of the molecular mechanism of the occurrence and development of PMOP is the top priority to grasp the disease course and develop targeted drugs.Currently,it is known that estrogen receptor activation can promote the differentiation of osteoblasts and inhibit the secretion of RANKL by osteoblasts.Inhibit osteoclast differentiation and promote osteoclast apoptosis.At the same time,it is found that the expression of PDZK1(PDZ domain-containing protein 1),a widely existing scaffold protein in the body,is positively correlated with estrogen level and is one of the downstream molecules of estrogen signal transduction.At present,most studies around PDZK1 focus on breast cancer,uric acid metabolism,and lipid metabolism,while its role in bone metabolism has not been reported.Therefore,the first part of the study aims to explore whether estrogen deficiency can cause the change of PDZK1 expression in bone tissue and whether the change of PDZK1 expression affects bone metabolism.【Methods】1.A three-month-old female C57BL/6J mouse model was constructed by oophorectomy.Bone density,trabecular structure and cortical thickness of bone were observed by micro-CT and Masson staining.Bone resorption marker CTX-1 was observed by serum comparison,and osteoclasts were compared by immunofluorescence staining of bone tissue to confirm successful modeling.The transcription and protein levels of PDZK1 in sham operation group and model group were compared.2.CRISPR-Cas9 technology was used to construct PDZK1whole-body knockout C57BL/6J mice.Western-blotting technique and immunohistochemistry were used to determine the knockout effect.3.Oophorectomy was performed on 3-month-old female mice with whole-body PDZK1 knockout to compare whether the absence of PDZK1 would aggravate the osteoporosis caused by estrogen reduction.【Results】1.Four months after oophorectomy in three-month-old female mice,the results of micro-CT showed that the femoral bone density of OVX mice decreased,the trabecular separation increased,and the bone cortex became thinner.Masson staining showed a decrease in bone trabecula and a large number of fat vacuoles in bone marrow of OVX mice.ELISA results showed that the content of serum bone resorption marker CTX-1 in OVX mice was significantly increased.Immunofluorescence results showed that osteoclasts increased significantly in epiphyseal area of OVX mice,accompanied by downregulation of PDZK1 expression.At the same time,the transcription level and protein expression level of PDZK1 in OVX mice bone tissue were down-regulated.2.Almost no PDZK1 expression was detected in kidney,liver and bone tissues of whole-body PDZK1 knockout mice.Compared with wild-type mice,PDZK1 knockout mice showed decreased trabecular bone volume,no difference in bone cortex,narrowed trabecular width and reduced degree of separation,but there was no statistical difference.At the same time,the serum CTX-1 of PDZK1 knockout mice was significantly increased,and the number of osteoclasts in epiphysis was significantly increased.The body length of PDZK1 knockout mice was slightly shorter than that of wild type mice,but there was no significant difference in body weight.The number of monocytes and neutrophils in whole blood samples of PDZK1 knockout mice was significantly increased.3.The bone mass of PDZK1 knockout mice decreased significantly 4 months after OVX.Compared with wild-type PMOP mice,bone trabeculae were thinner and more fat vacuoles appeared in bone marrow.【Conclusions】1.The expression of PDZK1 is decreased in PMOP mouse model.2.After PDZK1 knockout,osteoclast activity and bone resorption were increased in mice,but not as obviously as in estrogen-deficient mice.3.PDZK1 knockout aggravated bone loss in PMOP mice.These results indicate that PDZK1 positively regulates bone metabolism and transduces some functions of estrogen.PDZK1 deficiency is one of the main factors leading to PMOP.Part two: Study on the relationship between PDZK1 expression and osteoclast differentiation【Objects】After finding in the second part that the expression level of PDZK1 is correlated with the osteoclast differentiation process and the expression of NFATc1,this chapter aims to further explore whether PDZK1 regulates NFATc1 directly or indirectly.【Methods】1.Femurs of wild-type C57BL/6J mice aged three months were isolated,and primary bone marrow mononuclear cells/macrophages were extracted from the bone marrow.M-CSF and RANKL were used to induce osteoclasts.TRAP staining was performed at 0min(without inducible factors),5min,15 min,30min,60 min,120min and 3rd,5th and 7th days of induction differentiation in vitro to observe osteoclast formation,and protein collection and RNA extraction were performed to observe the expression changes of PDZK1 and transcription level changes.To detect whether PDZK1 is involved in osteoclast formation and activation.2.Primary bone marrow monocytes/macrophages were extracted from3-month-old female wild-type and PDZK1 knockout mice and cultured into osteoclasts in vitro.The formation of osteoclasts was observed by TRAP staining on the seventh day of induced differentiation.The formation of F-actin ring and bone absorption area were observed by induction differentiation on bone slices respectively.Proteins were collected and RNA was extracted to detect the levels of osteoclast-related transcription factors NFATc1,CTSK,MMP9 and Rank,so as to detect whether the loss of PDZK1 promoted the formation and activation of osteoclasts.3.Mouse monocyte/macrophage leukemia cell line RAW264.7 was cultured in vitro,and transfected with plasmid containing empty vector,si-PDZK1 and PDZK1 c DNA,M-CSF and RANKL were used to induce osteoclasts.The expression level of PDZK1 was detected by WB to determine the silenced/overexpressed effect.The osteoclast formation was observed by PCR to detect the transcription-related factors NFATc1,CTSK,MMP9 and Rank,and the osteoclast formation was observed by TRAP staining.F-actin ring formation and bone absorption area were observed on bone slices.Tunel staining was performed in the three groups and the resveratrol induced apoptosis group,and apoptosis-related protein levels were detected to observe the apoptosis of osteoclasts.To observe the effect of PDZK1 expression level on osteoclasts.【Results】1.Primary monocytes/macrophages began to fuse into multinucleated cells on the fifth day of osteoclast induction differentiation,and PDZK1 was highly expressed in monocytes/macrophages,but low expression was observed on the third day of osteoclast induction differentiation,and then increased.However,in cell lines,PDZK1 was only low in the initial stage,but increased gradually in the later stage.2.The primary mononuclear/macrophage cells of PDZK1 knockout mice could form osteoclasts with more nuclei,more F-actin rings,larger bone absorption area,and increased expressions of NFATc1,CTSK,and MMP9 during the induction and differentiation into osteoclasts in vitro.3.After PDZK1 silenced,osteoclast transcription-related factors of RAW264.7cells had higher transcription levels,more F-actin rings and larger bone absorption area;However,RAW264.7 cells with PDZK1 overexpression would be apoptotic and could not be further induced to become osteoclasts.【Conclusions】1.The expression of PDZK1 increased gradually during osteoclast fusion.2.Monocytes-macrophages with low expression of PDZK1 are more likely to fuse into osteoclasts.3.High expression of PDZK1 can induce monocyte/macrophage apoptosis.These results indicated that low expression of PDZK1 induced osteoclast activation and high expression promoted osteoclast apoptosis.Part Ⅲ: Study on the mechanism of PDZK1 inhibiting NFATc1 through calcium-modulating channels【Objects】After finding in the second part that the expression level of PDZK1 is correlated with the osteoclast differentiation process and the expression of NFATc1,this chapter aims to further explore whether PDZK1 regulates NFATc1 directly or indirectly.【Methods】1.The tissues of wild-type and knockout mice were sequenced by transcriptomics.The results showed that the pathways related to mineral and salt metabolism were up-regulated,including SLC8A1 gene encoding the sodium-calcium exchange NCX1.2.Osteoclast induction was performed on RAW264.7 cells transfected with empty vector,PDZK1 small interfering RNA,PDZK1 overexpression plasmid,and SLC8A1 small interfering RNA,respectively.Total protein was extracted on day 5 to observe the expression changes of calmodulator signal Ca MK-II and osteoclast differentiation regulator NFATc1.3.The calcium ion probe Fluo-4,AM was incubated with the above cells,and the cells were continuously observed with laser confocal and fluorescent enzyme labels.The fluorescence changes were continuously recorded within 10 minutes after taking pictures every 30 seconds.【Results】The transcriptomic results suggested that PDZK1 negatively regulated NCX1,and the structural prediction suggested that PDZK1 might bind to the calcium binding site of NCX1.The results of WB confirmed that PDZK1 negatively regulated NCX1,Ca MK-II,and NFATc1.PDZK1 negatively regulates calcium oscillation.【Conclusions】The absence of PDZK1 leads to increased expression of NCX1,calcium oscillation,activation of Ca MK-II,promotion of NFATc1 expression,and promotion of osteoclast differentiation.Part Ⅳ: The functional study of PDZK1 in osteoblast differentiation and RANKL secretion【Objects】Through the first,second and third part of the study,we clarified the mechanism of PDZK1 regulating osteoclasts through NFATc1.Since the activation of osteoclasts also requires the binding of RANKL to RANK,this chapter aims to further explore whether PDZK1 regulates the secretion of RANKL by osteoblasts in a direct or indirect way.【Methods】1.Femur was isolated from 3-month-old female wild-type C57BL/6J mice,and primary mesenchymal stem cells were extracted from bone marrow for osteogenic induction culture in vitro.At the 0,3,5 and 7 days of induction differentiation in vitro,ALP staining and WB were performed to observe the expression of BMP2 and observe the osteogenic differentiation,and proteins were collected and RNA was extracted to observe the expression and transcription level changes of PDZK1,so as to detect whether PDZK1 participated in the process of osteogenic differentiation.2.The serum of 3-month-old female wild-type and PDZK1 knockout mice was taken to detect the content and ratio of OPG and RANKL by ELISA and the primary mesenchymal stem cells were extracted respectively and induced and cultured into osteoblasts in vitro.On the 7th day of induced differentiation,ALP staining was performed and the expression of BMP2 was detected by WB to observe the early osteogenesis.The formation of calcium nodules was observed by alizarin red staining on the 14 th day of induction differentiation.At the same time,F-actin staining was performed to compare cell morphology to detect whether PDZK1 deletion affected osteogenic differentiation and mesenchymal stem cell function.3.Mouse osteoblast precursor cell line MC3T3-E1 from cranium roof was cultured in vitro,and transfected with plasmid containing empty vector,si-PDZK1 and PDZK1 c DNA,and then induced into osteoblasts by osteogenic induction medium.The silencing/overexpression effect of PDZK1 was determined by WB,and the expression levels of OPG and RANKL were detected by WB.The secretion of OPG and RANKL in cell medium was detected by ELISA.Osteoblast transcription-related factors BMP2,OPG,RANKL,OPN,ALP and RUNX2 were detected by PCR to observe the formation of osteoblasts.F-actin staining was used to compare cell morphology,so as to further observe the effect of PDZK1 expression level on osteoblast differentiation and the secretion of RANKL by osteoblasts.【Results】1.The expression of PDZK1 increased gradually during osteogenic differentiation.2.The serum OPG content of PDZK1 deletion mice decreased,and the serum RANKL content increased;OPG/RANKL ratio decreased;In MC3T3-E1 cell medium with PDZK1 silenced,the secretion of OPG remained unchanged,while the secretion of RANKL increased.MC3T3-E1 cells overexpressing PDZK1 decreased the secretion of OPG and increased the secretion of RANKL.3.The osteoblastic ability of MC3T3-E1 cells that were silted or lacking PDZK1 in the primary generation was decreased,and the osteoblastic ability of MC3T3-E1 cells that overexpressed PDZK1 was increased,while the morphological abnormalities of bone marrow mesenchymal stem cells that lacked PDZK1 in the primary generation were simultaneously observed.【Conclusions】PDZK1 is one of the proteins required for normal osteogenic differentiation.The lack of PDZK1 results in the abnormal morphology and decreased osteogenic ability of osteoblasts.Osteoblasts secreted more RANKL and less OPG,and the OPG/RANKL ratio decreased,which promoted osteoclast activation.Overexpression of PDZK1 promoted osteogenesis but also secreted more OPG,and the OPG/RANKL ratio tended to 1,without over-activation of osteoclasts.