The Role And Mechanism Of TMEM135-Mediated Mitochondrial Fission In Bone Marrow Mesenchymal Stem Cells In Osteoporosis

BackgroundOsteoporosis（OP）is a systemic disease characterized by a decrease in bone mineral density（BMD）and an elevated prevalence of fragility fractures.With the aging of the population,OP and its associated fractures have emerged as significant public health concerns impacting the elderly populace.Therefore,it is imperative to comprehend the etiology and mechanisms of OP,alongside identifying intervention targets.This will lay a theoretical foundation for treating OP and reducing the occurrence of complications such as osteoporotic fractures.Bone marrow mesenchymal stem cells（BMSCs）,as a class of multipotent progenitor cells originating from the bone marrow,play a pivotal role in bone development.Disrupting the equilibrium between osteogenic and adipogenic differentiation in BMSCs lead to disruption of bone homeostasis,causing bone loss,microstructural impairment,and ultimately resulting in osteoporosis.The mitochondrial quality control system,including mitochondrial biogenesis,mitochondrial dynamic,mitophagy,and mitochondrial transfer are critical for maintaining the homeostasis and differentiation trajectory of cells.Mitochondrial dynamics are key components of the mitochondrial quality control system,encompassing mitochondrial fission,fusion,movement,and distribution within cells.Studies have revealed notable altered mitochondrial morphology and dynamics during the differentiation of BMSCs.However,the intrinsic molecular mechanisms,as well as their roles and contributions to OP,remain inadequately understood.Transmembrane protein 135（TMEM135）,belonging to the transmembrane protein family,is located in peroxisomes,mitochondria,and cytosol.The involvement of TMEM135 in the regulation of mitochondrial fission has been verified.Previous bioinformatics has predicted a strong association between TMEM135 and the processes of osteogenesis and adipogenesis.Recent Genome-Wide Association Studies（GWAS）have identified TMEM135 as a novel candidate gene associated with BMD.However,the precise role of TMEM135 in OP is unclear.How TMEM135 regulates bone metabolism and whether this process involves mitochondrial dynamics remain to be elucidated.Objectives（1）Investigate the changes in mitochondrial dynamics during the process of osteogenic and adipogenic differentiation of BMSCs,and explore the impact of the regulation of mitochondrial fusion and fission on osteogenesis and adipogenesis,aiming to identify potential targets for mitochondrial dynamics in the regulation of BMSCs differentiation;（2）Explore the role of TMEM135 in bone development and the effects of TMEM135deficiency on osteogenic and adipogenic differentiation of BMSCs.Furthermore,we investigate the mechanism whereby TMEM135 modulates osteogenic differentiation of BMSCs via the regulation of mitochondrial fission;（3）Investigate the impact of intervening in TMEM135 expression and mitochondrial dynamics on bone in osteoporotic mouse models,providing a research foundation for gene-targeted therapy and mitochondrial dynamic therapy for osteoporosis.MethodsPartⅠ:Study on the mitochondrial dynamics during the differentiation of BMSCs（1）BMSCs were extracted from C57BL/6 mice and induced osteogenic and adipogenic differentiation in vitro.The degree of osteogenic differentiation was assessed through alkaline phosphatase staining and alizarin red staining.The degree of adipogenic differentiation was evaluated through Oil Red O staining and Bodipy staining.Changes in mitochondrial morphology in BMSCs were observed through Mito-tracker staining and transmission electron microscopy,and the expression changes of genes related to mitochondrial dynamics during the osteogenic and adipogenic differentiation of BMSCs were detected through Western-blot and RT-q PCR experiments;（2）Seahorse experiments were performed to measure the changes in oxygen consumption rate（OCR）,adenosine triphosphate（ATP）,and other metabolic levels during the osteogenic and adipogenic differentiation of BMSCs;（3）BMSCs were infected with overexpressed（OE）Dnm1l,Fis1,and Mfn2 genes lentiviruses to overexpress genes related to mitochondrial dynamics.Then the osteogenic and adipogenic differentiation abilities of BMSCs in each group were evaluated,respectively;（4）BMSCs were treated with mitochondrial fission promoter BC1618 and fusion promoter M1 agonist.Subsequently,alterations in the osteogenic and adipogenic differentiation abilities of BMSCs after treatment were observed.PartⅡ:Study of the influence of TMEM135 on osteogenic and adipogenic differentiation,and osteoporosis-related phenotypes caused by TMEM135 deficiency（1）Screening of differentially expressed genes（DEGs）from GSE35956 database（Transcriptome analyses of human primary osteoporosis and advanced donor age）and GSE80614 database（Gene expression analyses of the differentiating human BMSCs into adipocytes.）were performed.Subsequently,an intersection between the identified DEGs and the GWAS databases was extracted.Finally,genes of interest associated with mitochondrial fission and fusion functions（GO:0000266;GO:0008053）were extracted through a filtering process;（2）Immunofluorescence stainings were conducted on both bone tissue and cells.The expression levels of TMEM135 in spinous process bone tissue samples from OP patients and normal donors were assessed and compared through RT-q PCR and Western-blot analysis,aiming to elucidate the role of TMEM135 in bone metabolism;（3）The lentiviruses overexpressing Tmem135（OE-Tmem135）and short hairpin RNA targeting Tmem135（Sh-Tmem135）were constructed to establish stable cell lines with overexpression or knockdown of Tmem135 gene in BMSCs.The effects of TMEM135knockdown and overexpression on the osteogenic and adipogenic differentiation abilities of BMSCs were evaluated through Western-blot,RT-q PCR,and staining experiments;（4）The CRISPR/Cas9 technology was employed to construct a Tmem135 gene knockout C57BL/6 mouse model(Tmem135^-/-).The phenotypes of knockout mice and control mice were compared through experiments such as micro-CT,bone tissue staining,and calcium green double labeling.PartⅢ:Study on the mechanism underlying TMEM135 regulation of cell osteogenic differentiation via mitochondrial fission（1）Immunofluorescence,transmission electron microscopy,and OCR detection were conducted to assess the impacts of TMEM135 on mitochondrial dynamics and cellular energy metabolism;（2）Analysis of RNA-seq data was performed to explore mechanisms by which TMEM135 regulates mitochondrial fission;（3）Flow cytometry and immunofluorescence experiments were employed to assess the impacts of TMEM135 deficiency on intracellular calcium ion homeostasis;（4）Western-blot experiments and calcineurin（CALNA）activity assays were used to verify the effects of TMEM135-mediated Ca²⁺/Ca M/CALNA signaling pathway on the dephosphorylation of DRP1 protein at Serine 637.Interaction between CALNA and DRP1was investigated through co-immunoprecipitation experiments;（5）The calmodulin agonist CALP1 was employed to verify the role of TMEM135 in promoting osteogenic differentiation by activating the Ca²⁺/Ca M/CALNA signaling pathway.PartⅣ:Exploring strategies for the treatment of osteoporosis targeting TMEM135 and mitochondrial dynamics（1）The therapeutic effect of targeting mitochondrial dynamics on OP was assessed via intraperitoneal injection of a mitochondrial dynamic promoters.HE staining was performed to observe the histopathological changes in livers;（2）The mice ectopic osteogenesis model was established to evaluate the impact of overexpressing TMEM135;（3）Adeno-associated virus（AAV）overexpressing the Tmem135 gene were constructed.The osteoporotic mice were treated through injections of AAV into femoral bone marrow cavity.The efficacy of targeting TMEM135 for bone loss was evaluated using micro-CT and histological examination.ResultsPartⅠ:Study on the mitochondrial dynamics during the differentiation of BMSCs（1）Mito-tracker fluorescence staining and transmission electron microscopy showed distinctly morphological changes in mitochondria during BMSC differentiation into osteoblasts and adipocytes.Western-blot and RT-q PCR results suggested that mitochondrial dynamics undergo fusion at the early stage while shifting toward fission at the late stage during osteogenic differentiation.Conversely,mitochondrial dynamics are inclined to fusion during adipogenic differentiation;（2）Cellular energy metabolism assessment showed that OCR and ATP levels gradually increased during early osteogenic differentiation,peaking on the seventh day;OCR and ATP levels gradually increased during adipogenic differentiation;（3）Regulation of genes related to mitochondrial dynamics affected the BMSCs osteogenic and adipogenic differentiation ability.Overexpression of Dnm1l and Fis1 genes upregulated osteogenic differentiation,whereas overexpression of the Fis1 gene attenuated adipogenic differentiation.Overexpression of the Mfn2 gene promoted adipogenic differentiation.PartⅡ:Study of the influence of TMEM135 on osteogenic and adipogenic differentiation,and osteoporosis-related phenotypes caused by TMEM135 deficiency（1）The Tmem135 gene was screened through GSE35956,GSE80614,GWAS databases,and the gene sets related to mitochondrial fission and fusion functions（GO:0000266,GO:0008053）;（2）TMEM135 expression was widespread in bone tissue and has high expression in mitochondria and cytosol.The TMEM135 expression was upregulated during BMSC osteogenic differentiation.The expression level of TMEM135 in the bone tissue of OP patients is significantly decreased compared to that of normal individuals;（3）Knockdown of TMEM135 inhibited osteogenic differentiation and enhanced adipogenic differentiation ability.Upregulation of TMEM135 promoted osteogenic differentiation;（4）Tmem135^-/-mice exhibited osteoporotic phenotypes,which are characterized by reduced bone mass and increased white adipose tissue.The osteoporotic phenotypes were progressively aggravated with age.PartⅢ:Study on the mechanism underlying TMEM135 regulation of cell osteogenic differentiation via mitochondrial fission（1）Mito-tracker fluorescence staining and transmission electron microscopy revealed that deletion of the Tmem135 gene resulted in impairment of mitochondrial fission in the late stage of BMSC osteogenic differentiation,leading to suppression of oxidative phosphorylation in mitochondria;（2）RNA-seq indicated that deletion of TMEM135 led to negative regulation of the calcium signaling pathway.Flow cytometry assessment suggested that TMEM135deficiency disrupted intracellular calcium ions homeostasis in BMSCs and inhibited the upregulation of intracellular calcium ions following osteogenic differentiation;（3）Immunoprecipitation and Western-blot results suggested that TMEM135 could promote the dephosphorylation of DRP1 at serine 637 by calcineurin,thereby promoting the translocation of DRP1 to the outer membrane of mitochondria and triggering mitochondrial fission;（4）CALP1 rescued the osteogenic ability of Tmem135^-/-primary BMSCs by restoring mitochondrial fission in the（middle-to-late stage）osteogenic differentiation.PartⅣ:Exploring strategies for the treatment of osteoporosis targeting TMEM135 and mitochondrial dynamics（1）Intraperitoneal injection of the mitochondrial fission promoter BC1618 improved bone loss in osteoporosis model mice.However,HE stainings suggested that the mice liver was damaged to a certain extent;（2）Hydroxyapatite scaffolds with BMSCs overexpressing Tmem135 promoted subcutaneous ectopic ossification in NSG mice;（3）Micro-CT and histological examination results indicated that AAV-mediated TMEM135 was effectively expressed in bone tissue.Injection into the femoral bone marrow cavity with Tmem135 overexpression AAV significantly improved OVX-induced osteoporotic bone loss.ConclusionsMitochondrial dynamics underwent distinct alterations during the osteogenic and adipogenic differentiation of BMSCs.We identify,for the first time,TMEM135 as being an upstream gene regulating mitochondrial fission during BMSCs differentiation.TMEM135plays a crucial role in maintaining the equilibrium of osteogenesis and adipogenesis of BMSCs.Deletion of TMEM135 in mice leads to osteoporotic phenotypes by inhibiting osteogenic differentiation and enhancing adipogenic differentiation of BMSCs.TMEM135increases the intracellular concentration of calcium ions,activates the Ca²⁺/Ca M/CALNA signaling pathway,and dephosphorylates DRP1 at the Ser637 through CALNA,thereby triggering mitochondrial fission.Targeting TMEM135 and mitochondrial dynamics could enhance the osteogenic differentiation ability of BMSCs and improve bone loss in osteoporotic model mice.This study provides a foundation and new insights for gene and mitochondrial dynamics-targeted therapy for osteoporosis.