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Principal Investigator: Reyad A Elbarbary
Organization: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
Fiscal Year: 2019
Award: $395,786
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
Abstract
Impaired or delayed fracture healing is a clinical problem that affects >1.5 million people in the US annually;
obesity and associated type 2 diabetes (T2D) are significant and independent risk factors in this context.
Nearly 34% of the US population is obese, and the number is projected to climb significantly in the coming
decade. Therefore, the incidence of obesity/T2D-associated impaired fracture healing will be a growing
concern. Despite these sobering statistics, the molecular basis for delayed healing in obesity/T2D remains
unknown and begs investigation. Recently, it has been established that impaired fracture healing in the diet-
induced obesity (DIO) mouse model, which is an established model of obesity and hyperglycemia, is
accompanied by an increased number of adipocytes within fracture callus. We followed up on these studies to
discover that Staphylococcal nuclease and tudor domain-containing 1 (Tudor-SN, abbreviated as TSN)
promotes adipogenesis in murine primary bone marrow-derived mesenchymal stem cells (BMSCs), as well as
in mouse 3T3-L1 and human HprAD preadipocytes, via degrading particular anti-adipogenic microRNAs
(miRNAs), including two key miRNAs that inhibit the expression of peroxisome proliferator-activated receptor
gamma (PPARg), the master regulator of adipogenesis. Remarkably, we also found that TSN expression is
focally elevated within the callus of DIO mice compared to lean mice, co-localizing with PPARg in the woven-
bone lining cells, and occurring at time points immediately preceding the adipocyte bloom. Downregulating
callus TSN via local delivery of a chemically modified TSN siRNA inhibited adipogenesis and enhanced
mineralized callus formation in DIO mice. According to these preliminary data, we propose the central
hypothesis that TSN is a key molecular mediator of the delayed bone healing that occurs in obesity/T2D. To
test this hypothesis, we propose to execute two Specific Aims. In the first Specific Aim, we will elucidate the
role of TSN as a regulator of BMSCs differentiation and a mediator of adipogenesis that promotes the turnover
of anti-adipogenic miRNAs. We will use RNA-seq, miR-seq, and RT-qPCR to study the effect of TSN knockout
on the mRNA and miRNA pools in primary BMSCs isolated from wild-type and TSN knockout mice. In the
second Specific Aim, we will characterize TSN function in delayed bone healing in DIO mice. We will first
compare the expression levels of fracture healing-associated genes at various stages of healing in lean and
DIO mice. Comparisons will be performed on the transcriptomic level using RNA-seq, RT-qPCR, and multiplex
fluorescence in situ hybridization, and on the protein level using immunofluorescence staining. We will also
compare the expression levels of miRNAs using miR-seq and RT-qPCR. Finally, we will study the impact of
delivering a TSN siRNA to the fracture callus on different stages of healing in lean and DIO mice. Completion
of the proposed experiments will enable us to define the role of TSN and its target miRNAs in the process of
fracture repair and implicate TSN in obesity/T2D-associated impaired fracture healing.
Terms: <Active Follow-up><Address><Adipocytes><Adipose Cell><Adult-Onset Diabetes Mellitus><Affect><Autoregulation><Basal Transcription Factor><Basal transcription factor genes><Biology><Bone Formation><Bone Marrow><Bone Marrow Reticuloendothelial System><Bone callus><Bony Callus><Bundle Bone><Callus><Cell Body><Cell Differentiation><Cell Differentiation process><Cells><Chemicals><Clinical><Complex><Data><Defect><Development><Diet><Differential Gene Expression><FISH Technic><FISH Technique><FISH analysis><FISH assay><Fat Cells><Fats><Fatty acid glycerol esters><Fluorescence In Situ Hybridization><Fluorescent in Situ Hybridization><Fracture><Fracture Healing><Gene Expression><General Transcription Factor Gene><General Transcription Factors><Genes><Grant><Healing abnormal><Healing delayed><Histologic Technics><Histologic Techniques><Histological Technics><Histological Techniques><Homeostasis><Human><Hydrogels><Hyperglycemia><Immature Bone><Immunofluorescence><Immunofluorescence Immunologic><Impaired healing><Impaired tissue repair><Impaired wound healing><Impairment><In Vitro><Incidence><Investigation><KO mice><Ketosis-Resistant Diabetes Mellitus><Knock-out><Knock-out Mice><Knockout><Knockout Mice><Leanness><Lipocytes><Mammalian Cell><Marrow><Mature Lipocyte><Mature fat cell><Maturity-Onset Diabetes Mellitus><Measures><Mediating><Mediator><Mediator of Activation><Mediator of activation protein><Mesenchymal Differentiation><Mesenchymal Progenitor Cell><Mesenchymal Stem Cells><Messenger RNA><Mice><Mice Mammals><Micro RNA><MicroRNA Expression Profiling><MicroRNAs><Micrococcal Nuclease><Minerals><Modeling><Modern Man><Molecular><Murine><Mus><Musculoskeletal><NIDDM><NIH><National Institutes of Health><Non-Insulin Dependent Diabetes><Non-Insulin-Dependent Diabetes Mellitus><Noninsulin Dependent Diabetes><Noninsulin Dependent Diabetes Mellitus><Nonlamellar Bone><Null Mouse><Obese Mice><Obesity><Osteogenesis><P-30 Protein><P30><P30 Protein><PPAR gamma><PPAR-γ><PPARgamma><PPARγ><Pathologic><Pathology><Pathway interactions><Peroxisome Proliferative Activated Receptor Gamma><Peroxisome Proliferator-Activated Receptor gamma><Peroxisome Proliferator-Activated Receptor γ><Personalized medical approach><Phase><Phenotype><Physiological Homeostasis><Play><Population><Process><Proteins><RNA Seq><RNA sequencing><RNAseq><Regulation><Reporting><Research Resources><Resources><Risk Factors><Role><Science><Short interfering RNA><Site><Slow-Onset Diabetes Mellitus><Small Interfering RNA><Stable Diabetes Mellitus><Staining method><Stains><Staphylococcal Nuclease><T2 DM><T2D><T2DM><TNase><Testing><Thermonuclease><Thermostable Nuclease><Thiazolidinedione Receptor><Thinness><Time><Tissue-Specific Differential Gene Expression><Tissue-Specific Gene Expression><Transcription Factor Proto-Oncogene><Transcription factor genes><Type 2 Diabetes Mellitus><Type 2 diabetes><Type 2 diabetic><Type II Diabetes Mellitus><Type II diabetes><Type II diabetic><United States National Institutes of Health><Work><Woven Bone><abnormal tissue repair><active followup><adipogenesis><adiposity><adult onset diabetes><bone fracture><bone healing><bone tissue formation><corpulence><corpulency><corpulentia><delayed wound healing><developmental><diabetic patient><dietary><experiment><experimental research><experimental study><follow up><follow-up><followed up><followup><genome scale><genome wide analysis><genome wide studies><genome-wide><genome-wide analysis><genome-wide identification><genomewide><global miRNA profiling><healing><high reward><hyperglycemic><individualized approach><ketosis resistant diabetes><lipid biosynthesis><lipogenesis><mRNA><maturity onset diabetes><miRNA><miRNA expression profiling><miRNA sequencing><miRNA-seq><miRNAs><micro RNA expression profiling><microRNA sequencing><mouse model><multipotent cell><murine model><nano particle><nano-sized particle><nanoparticle><nanosized particle><new drug treatments><new drugs><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutics><new therapy><new therapy approaches><next generation therapeutics><novel><novel drug treatments><novel drugs><novel therapeutic approach><novel therapeutic intervention><novel therapeutics><novel therapy><novel therapy approach><nuclease><ob/ob mouse><obese><obese people><obese person><obese population><osteogenic><pathway><personalized approach><precision approach><ranpirnase><repair><repaired><role model><siRNA><social role><statistics><stem cell differentiation><tailored approach><transcription factor><transcriptome sequencing><transcriptomics><type 2 DM><type II DM><type two diabetes>