Differential effects of Wnt-ß-catenin signaling in Purkinje cells and Bergmann glia in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease characterized by progressive ataxia and degeneration of specific neuronal populations, including Purkinje cells (PCs) in the cerebellum. Previous studies have demonstrated a critical role for various evolutionarily conserved signaling pathways in cerebellar patterning, such as the Wnt-ß-catenin pathway; however, the roles of these pathways in adult cerebellar function and cerebellar neurodegeneration are largely unknown. In this study, we found that Wnt-ß-catenin signaling activity was progressively enhanced in multiple cell types in the adult SCA1 mouse cerebellum, and that activation of this signaling occurs in an ataxin-1 polyglutamine (polyQ) expansion-dependent manner. Genetic manipulation of the Wnt-ß-catenin signaling pathway in specific cerebellar cell populations revealed that activation of Wnt-ß-catenin signaling in PCs alone was not sufficient to induce SCA1-like phenotypes, while its activation in astrocytes, including Bergmann glia (BG), resulted in gliosis and disrupted BG localization, which was replicated in SCA1 mouse models. Our studies identify a mechanism in which polyQ-expanded ataxin-1 positively regulates Wnt-ß-catenin signaling and demonstrate that different cell types have distinct responses to the enhanced Wnt-ß-catenin signaling in the SCA1 cerebellum, underscoring an important role of BG in SCA1 pathogenesis.

Congenital heart defects in CTNNB1 syndrome: Raising clinical awareness.

CTNNB1 [OMIM *116806] encodes ß-catenin, an integral part of the cadherin/catenin complex, which functions as effector of Wnt signaling. CTNNB1 is highly expressed in brain as well as in other tissues, including heart. Heterozygous CTNNB1 pathogenic variations are associated with a neurodevelopmental disorder characterized by spastic diplegia and visual defects (NEDSDV) [OMIM #615075], featuring psychomotor delay, intellectual disability, behavioral disturbances, movement disorders, visual defects and subtle facial and somatic features. We report on a new series of 19 NEDSDV patients (mean age 10.3 years), nine of whom bearing novel CTNNB1 variants. Notably, five patients showed congenital heart anomalies including absent pulmonary valve with intact ventricular septum, atrioventricular canal with hypoplastic aortic arch, tetralogy of Fallot, and mitral valve prolapse. We focused on the cardiac phenotype characterizing such cases and reviewed the congenital heart defects in previously reported NEDSDV patients. While congenital heart defects had occasionally been reported so far, the present findings configure a higher rate of cardiac anomalies, suggesting dedicated heart examination to NEDSDV clinical management.

MicroRNA-590-5p functions as a tumor suppressor in breast cancer conferring inhibitory effects on cell migration, invasion, and epithelial-mesenchymal transition by downregulating the Wnt-ß-catenin signaling pathway.

Breast cancer remains one of the foremost primary causes of female morbidity and mortality worldwide. During the current study, the effect of miR-590-5p and paired-like homeodomain transcription factor 2 (PITX2) on proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) of human breast cancer via the Wnt-ß-catenin signaling pathway was investigated. Breast cancer-related genes and related signaling pathways were obtained from KEGG database. The PITX2 regulatory microRNA was predicted. To define the contributory role by which miR-590-5p influences the progression of breast cancer, the interaction between miR-590-5p and PITX2 was explored; the proliferation, invasion, and migration abilities as well as the tumor growth and metastasis in nude mice were detected following the overexpression or silencing of miR-590-5p. PITX2 was determined to share a correlation with breast cancer and miR-590-5p was selected for further analysis. PITX2, Wnt-1, ß-catenin, N-cadherin, and vimentin all displayed higher levels, while miR-590-5p and E-cadherin expression were lower among breast cancer tissues than in the adjacent normal tissue. After overexpression of miR-590-5p or si-PITX2, the expression of E-cadherin was markedly increased, decreases in the expression of Wnt-1, ß-catenin, N-cadherin, and vimentin, as well as inhibited cell proliferation, invasion, migration, metastasis, and EMT were observed. This study provides evidence suggesting that the transfection of overexpressed miR-590-5p can act to alleviate the effects of breast cancer demonstrating an ability to inhibit the processes of cell proliferation, migration, and invasion as well as EMT by suppressing the expression of PITX2 and activation of the Wnt-ß-catenin pathway.

Specific knockdown of WNT8b expression protects against phosphate-induced calcification in vascular smooth muscle cells by inhibiting the Wnt-ß-catenin signaling pathway.

In the last 10 years, the prevalence, significance, and regulatory mechanisms of vascular calcification (VC) have gained increasing recognition. The aim of this study is to explore the action of WNT8b in the development of phosphate-induced VC through its effect on vascular smooth muscle cells (VSMCs) in vitro by inactivating the Wnt-ß-catenin signaling pathway. To explore the effect of WNT8b on the Wnt-ß-catenin signaling pathway and VC in vitro, ß-glycerophosphate (GP)-induced T/G HA-VSMCs were treated with small interfering RNA against WNT8b (Si-WNT8b), Wnt-ß-catenin signaling pathway activator (LiCl) and both, respectively. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine the messenger RNA and protein levels of WNT8b, α-smooth muscle actin (α-SMA), calcification-associated molecules, and molecules related to the Wnt signaling pathway. The TOP/FOP-Flash reporter assay was performed to detect the transcription activity mediated by ß-catenin. Si-WNT8b reduced calcium deposition and the activity of alkaline phosphatase (ALP), increased the α-SMA level, and decreased bone morphogenetic protein 2, Pit1, MSX2, and Runt-related transcription factor 2 levels, whereas stimulation of LiCl worsened ß-GP-induced calcium deposition, increased the activity of ALP, and reduced the α-SMA expression level. Si-WNT8b reduced the levels of WNT8b, frizzled-4, ß-catenin, phospho-GSK-3ß (p-GSK-3ß), and cyclin-D, whereas it increased the levels of p-ß-catenin and GSK-3ß, indicating that si-WNT8b could alter the Wnt-ß-catenin signaling pathway and thus hamper the VC in T/G HA-VSMC, which was further demonstrated by the TOP/FOP-Flash assay and detection of the ß-catenin expression level in the nucleus. Altogether, we conclude that WNT8b knockdown terminates phosphate-induced VC in VSMCs by inhibiting the Wnt-ß-catenin signaling pathway.

Analysis of the transcriptomic features of microsatellite instability subtype colon cancer.

BACKGROUND: Programmed cell death protein 1(PD-1) blocking antibodies have been used to enhance immunity in solid tumors and achieve durable clinical responses with an acceptable safety profile in multiple types of cancer. However, only a subset of patients could benefit from PD-1 blockade therapy. Prognostic information including PD-1 ligand (PD-L1) expression, IFN-γ expression signature, tumor mutational burden, and microsatellite instability (MSI) have been evaluated for patients who are selected to receive immune checkpoint therapeutic treatment. Yet the relationship of those biomarkers in determining immune checkpoint therapy is largely unknown. METHODS: Immune-profiles of MSI subtype colon cancer were identified from integrating published MSI associated gene expression data. The enriched pathways and transcription factors were analyzed by GSEA assay. The infiltrations of immune cell types into MSI subtype colon cancer tissues were determined by CIBESORT assay. RESULTS: In the MSI subtype colon cancer patients, PD-L1, IFN-γ and IFN-γ associated genes are highly expressed. And all those genes are favorable effects in colon cancer progress. In addition, we find that Wnt-ß-catenin and TGFß signaling pathways which are two important factors inhibiting PD-1 checkpoint blockade therapy are negatively related with MSI status. We also identify that the immune-profiles in MSI subtype colon cancer are contributed by M1 macrophage infiltration in the tumor environment. CONCLUSIONS: Our results provide the detailed underlying mechanisms of MSI subtype cancer patients are sensitive to PD-1 checkpoint blockade.

Factors associated with serum soluble inhibitors of Wnt-ß-catenin signaling (sclerostin and dickkopf-1) in patients undergoing peritoneal dialysis.

AIM: Sclerostin and dickkopf-1 (Dkk-1) are soluble inhibitors of Wnt-ß-catenin signaling and are involved in decreased bone formation and bone volume in patients with various bone diseases. The clinical characteristics of sclerostin and Dkk-1 and their impacts on mineral and bone metabolism remain undetermined in patients undergoing peritoneal dialysis (PD). METHODS: This cross-sectional study investigated the association between serum sclerostin and Dkk-1 levels and mineral disorders in 74 outpatients under PD treatment. Levels of sclerostin and Dkk-1 in serum, urine, and peritoneal dialysate were determined using enzyme-linked immunosorbent assay kits. The associations between serum sclerostin and Dkk-1 levels and biochemical parameters were evaluated by linear regression analyses. RESULTS: Median serum sclerostin and Dkk-1 levels were 138 pmol/L (range, 98.3-195.9 pmol/L) and 38.8 pmol/L (range, 28.5-47.1 pmol/L), respectively. Both sclerostin and Dkk-1 were excreted into urine and peritoneal dialysate. Multivariable linear regression analyses showed that serum sclerostin level was significantly associated with age, sex, parathyroid hormone level, and renal Kt/V. In contrast, serum Dkk-1 level was associated with platelet count and serum fibroblast growth factor 23 level but not with any of the bone metabolic markers. CONCLUSION: Serum sclerostin was associated with serum intact parathyroid hormone, while Dkk-1 was associated with serum fibroblast growth factor 23 in patients undergoing PD. The utility of determining soluble Wnt-ß-catenin inhibitors levels in patients undergoing PD requires further investigation.

Genetics of osteoporosis.

Osteoporosis is a skeletal disease characterized by low bone mineral density (BMD) and microarchitectural deterioration of bone tissue, which increases susceptibility to fractures. BMD is a complex quantitative trait with normal distribution and seems to be genetically controlled (in 50-90% of the cases), according to studies on twins and families. Over the last 20 years, candidate gene approach and genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) that are associated with low BMD, osteoporosis, and osteoporotic fractures. These SNPs have been mapped close to or within genes including those encoding nuclear receptors and WNT-ß-catenin signaling proteins. Understanding the genetics of osteoporosis will help identify novel candidates for diagnostic and therapeutic targets.

Single-Exon Deletions of ZNRF3 Exon 2 Cause Congenital Adrenal Hypoplasia.

CONTEXT: Primary adrenal insufficiency (PAI) is a life-threatening condition characterized by the inability of the adrenal cortex to produce sufficient steroid hormones. E3 ubiquitin protein ligase zinc and ring finger 3 (ZNRF3) is a negative regulator of Wnt/ß-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/ß-catenin signaling via binding and removal of ZNRF3 from the cell surface. OBJECTIVE: This work aimed to explore a novel genetic form of PAI. METHODS: We analyzed 9 patients with childhood-onset PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. To examine the functionality of the identified single-exon deletions of ZNRF3 exon 2, we performed three-dimensional (3D) structure modeling and in vitro functional studies. RESULTS: We identified various-sized single-exon deletions encompassing ZNRF3 exon 2 in 3 patients who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the 3 distinct single-exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein (ΔEx2-ZNRF3). Based on 3D structure modeling, we predicted that interaction between ZNRF3 and RSPO1 would be disturbed in ΔEx2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/ß-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/ß-catenin signaling was attenuated in cells expressing ΔEx2-ZNRF3 as compared with those expressing wild-type ZNRF3. CONCLUSION: We provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/ß-catenin signaling by ΔEx2-ZNRF3.

Regeneration capability of neonatal lung-derived decellularized extracellular matrix in an emphysema model.

Impaired and limited alveolar regeneration upon injury advances pulmonary disorders and irreversibly affects millions of people worldwide. Adult mammals do not have a strong potential to regenerate functional lung tissues, while neonatal lungs robustly proliferate and regenerate the functional tissue within a week of birth upon injury. The differential composition of the extracellular matrix (ECM) of neonatal tissues favors cellular proliferation and migration, fostering lung regeneration. Regardless, conventional ECM therapies employ adult-derived tissues. Therefore, the potential differences in regenerative properties of adult and neonatal lung ECM were investigated using in vitro and in vivo lung emphysema model. Decellularization of the neonatal and adult lungs was performed using freeze-thaw cycle method. Decellularization process was structurally characterized using SEM and immunostaining. In vitro treatment of neonatal lung-derived ECM (NECM) significantly enhanced the cellular migration and proliferation compared to adult-lung derived ECM (AECM) treated cigarette smoke-extract (CSE)-stimulated A549 cells. Following the administration of AECM and NECM, we observed a significant decline in emphysematous features and an improvement in lung functions in NECM group. NECM treatment increased the ratio of HOPX+/SpC+ cells with an active proliferation in SpC+ cells shown by colocalization of SpC+/Ki67+ and SpC+/Brdu+ cells. Moreover, NECM treatment activated the Neureguline-1/Erbb2 signaling and fostered a regenerative environment by upregulating the expression of regenerative genes including FGF, WNTs and AXIN-2 as compared to AECM treatment. Our findings suggested the potential utilization of NECM as novel therapeutics in regenerative medicine, deviating from the conventional application of adult-derived ECM treatments in pre-clinical and clinical research.

PORCN promotes hepatocellular carcinogenesis via Wnt/ß-catenin-dependent manner.

Background: Porcupine O-acyltransferase (PORCN), a membrane-bound O-acyltransferase, is crucial in Wnt ligand palmitoylation. However, the roles of PORCN in the development of hepatocellular carcinoma (HCC) remain unknown. Methods: Western blot, real-time quantitative polymerase chain reaction (RT-qPCR) assays, and The Cancer Genome Atlas (TCGA) database were used to study the expression and prognostic values of PORCN in patients with HCC. Following this, Cell Counting Kit-8 (CCK-8), wound-healing tests, Transwell assay, and a xenograft mouse model were employed to examine the effect of PORCN on HCC cells. Finally, the underlying molecular mechanisms involved in cell proliferation and migration caused by PORCN were identified. Results: The protein and messenger RNA (mRNA) levels of PORCN in HCC tissues were higher than those of adjacent normal tissues. The analysis of TCGA database indicated that patients with higher PORCN expression had a lower overall survival (OS) rate. Overexpression of PORCN could promote the proliferation and migration abilities of HCC cells both in vitro and in vivo. Gene set enrichment analysis (GSEA) showed that the effect of PORCN on the biological characteristics of HCC cells mainly centered on the Wnt-ß-catenin signaling pathway. Mechanically, immunofluorescence staining and subcellular protein fraction assays showed that PORCN could induce epithelial-mesenchymal transition (EMT) by promoting the translocation of ß-catenin from the cytoplasm to nucleus, ultimately promoting the progression of HCC. Conclusions: The findings of this study suggest that PORCN can promote HCC cell proliferation and migration by stimulating the Wnt-ß-catenin signaling pathway. Therefore, PORCN may be a promising therapeutic target for HCC.

Effects of Wnt-ß-Catenin Signaling and Sclerostin on the Phenotypes of Rat Pheochromocytoma PC12 Cells.

Pheochromocytomas and paragangliomas (PPGLs) are classified into 3 major categories with distinct driver genes: pseudohypoxia, kinase signaling, and Wnt-altered subtypes. PPGLs in the Wnt-altered subtype are sporadic and tend to be aggressive with metastasis, where somatic gene fusions affecting mastermind-like 3 (MAML3) and somatic mutations in cold shock domain containing E1 (CSDE1) cause overactivation of Wnt-ß-catenin signaling. However, the relation between Wnt-ß-catenin signaling and the biological behavior of PPGLs remains unexplored. In rat pheochromocytoma PC12 cells, Wnt3a treatment enhanced cell proliferation and suppressed mRNA expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis, and dopamine secretion. We identified the expression of sclerostin in PC12 cells, which is known as an osteocyte-derived negative regulator for Wnt signaling-driven bone formation. Inhibition of endogenous Wnt pathway by XAV939 or sclerostin resulted in attenuated cell proliferation and increased TH expression. Furthermore, Wnt3a pretreatment suppressed bone morphogenetic protein (BMP)-induced Smad1/5/9 phosphorylation whereas BMPs enhanced sclerostin expression in PC12 cells. In the Wnt-altered subtype, the increased Wnt-ß-catenin pathway may contribute the aggressive clinical behavior with reduced catecholamine production. Furthermore, upregulated expression of sclerostin by BMPs may explain the osteolytic metastatic lesions observed in metastatic PPGLs.

ASPM promotes glioblastoma growth by regulating G1 restriction point progression and Wnt-ß-catenin signaling.

Increasing evidence has indicated that the disorganized expression of certain genes promotes tumour progression. In this study, we elucidate the potential key differentially expressed genes (DEGs) between glioblastoma (GBM) and normal brain tissue by analysing three different mRNA expression profiles downloaded from the Gene Expression Omnibus (GEO) database. DEGs were sorted, and key candidate genes and signalling pathway enrichments were analysed. In our analysis, the highest fold change DEG was found to be abnormal spindle-like microcephaly associated (ASPM). The ASPM expression pattern from the database showed that it is highly expressed in GBM tissue, and patients with high expression of ASPM have a poor prognosis. Moreover, ASPM showed aberrantly high expression in GBM cell lines. Loss-of-function assay indicated that ASPM enhances tumorigenesis in GBM cells in vitro. Xenograft growth verified the oncogenic activity of ASPM in vivo. Furthermore, downregulation of ASPM could arrest the cell cycle of GBM cells at the G0/G1 phase and attenuate the Wnt/ß-catenin signalling activity in GBM. These data suggest that ASPM may serve as a new target for the therapeutic treatment of GBM.

An update on the role of RANKL-RANK/osteoprotegerin and WNT-ß-catenin signaling pathways in pediatric diseases.

BACKGROUND: Bone remodeling is a lifelong process due to the balanced activity of osteoclasts (OCs), the bone-reabsorbing cells, and osteoblasts (OBs), and the bone-forming cells. This equilibrium is regulated by numerous cytokines, but it has been largely demonstrated that the RANK/RANKL/osteoprotegerin and Wnt/ß-catenin pathways play a key role in the control of osteoclastogenesis and osteoblastogenesis, respectively. The pro-osteoblastogenic activity of the Wnt/ß-catenin can be inhibited by sclerostin and Dickkopf-1 (DKK-1). RANKL, sclerostin and DKKs-1 are often up-regulated in bone diseases, and they are the target of new monoclonal antibodies. DATA SOURCES: The authors performed a systematic literature search in PubMed and EMBASE to June 2018, reviewed and selected articles, based on pre-determined selection criteria. RESULTS: We re-evaluated the role of RANKL, osteoprotegerin, sclerostin and DKK-1 in altered bone remodeling associated with some inherited and acquired pediatric diseases, such as type 1 diabetes mellitus (T1DM), alkaptonuria (AKU), hemophilia A, osteogenesis imperfecta (OI), 21-hydroxylase deficiency (21OH-D) and Prader-Willi syndrome (PWS). To do so, we considered recent clinical studies done on pediatric patients in which the roles of RANKL-RANK/osteoprotegerin and WNT-ß-catenin signaling pathways have been investigated, and for which innovative therapies for the treatment of osteopenia/osteoporosis are being developed. CONCLUSIONS: The case studies taken into account for this review demonstrated that quite frequently both bone reabsorbing and bone deposition are impaired in pediatric diseases. Furthermore, for some of them, bone damage began in childhood but only manifested with age. The use of denosumab could represent a valid alternative therapeutic approach to improve bone health in children, although further studies need to be carried out.

LRP5, Bone Density, and Mechanical Stress: A Case Report and Literature Review.

The Wnt-ß-catenin pathway receptor, low-density lipoprotein receptor-related protein 5 (LRP5), is a known regulator of bone mineral density. It has been hypothesized that specific human polymorphisms in LRP5 impact bone density, in part, by altering the anabolic response of bone to mechanical loading. Although experiments in animal models support this hypothesis, there is limited evidence that LRP5 polymorphisms can alter the anabolic response of bone to mechanical loading in humans. Herein, we report a young male who harbors a rare LRP5 missense mutation (A745V) and who provides potential proof of principle for this mechanotransduction hypothesis for low bone density. The subject had no history of fractures until age 18, a year into a career in competitive distance running. As he continued to run over the following 2 years, his mileage threshold to fracture steadily and rapidly decreased until he was diagnosed with severe osteoporosis (lumbar spine BMD Z-score of -3.2). By contextualizing this case within the existing LRP5 and mechanical stress literature, we speculate that this represents the first documented case of an individual in whom a genetic mutation altered the anabolic response of bone to mechanical stress in a manner sufficient to contribute to osteoporosis.

Downregulation of the Repressor Element 1-Silencing Transcription Factor (REST) Is Associated with Akt-mTOR and Wnt-ß-Catenin Signaling in Prion Diseases Models.

Prion diseases are a group of infectious diseases characterized by multiple neuropathological changes, yet the mechanisms that preserve function and protect against prion-associated neurodegeneration are still unclear. We previously reported that the repressor element 1-silencing transcription factor (REST) alleviates neurotoxic prion peptide (PrP106-126)-induced toxicity in primary neurons. Here we confirmed the findings of the in vitro model in 263K infected hamsters, an in vivo model of prion diseases and further showed the relationships between REST and related signaling pathways. REST was depleted from the nucleus in prion infected brains and taken up by autophagosomes in the cytoplasm, co-localizing with LC3-II. Importantly, downregulation of the Akt-mTOR and at least partially inactivation of LRP6-Wnt-ß-catenin signaling pathways correlated with the decreased levels of REST in vivo in the brain of 263K-infected hamsters and in vitro in PrP106-126-treated primary neurons. Overexpression of REST in primary cortical neurons alleviated PrP106-126 peptide-induced neuronal oxidative stress, mitochondrial damage and partly inhibition of the LRP6-Wnt-ß-catenin and Akt-mTOR signaling. Based on our findings, a model of REST-mediated neuroprotection in prion infected animals is proposed, with Akt-mTOR and Wnt-ß-catenin signaling as the key pathways. REST-mediated neuronal survival signaling could be explored as a viable therapeutic target for prion diseases and related neurodegenerative diseases.

REST alleviates neurotoxic prion peptide-induced synaptic abnormalities, neurofibrillary degeneration and neuronal death partially via LRP6-mediated Wnt-ß-catenin signaling.

Prion diseases are a group of infectious neurodegenerative diseases characterized by multiple neuropathological hallmarks including synaptic damage, spongiform degeneration and neuronal death. The factors and mechanisms that maintain cellular morphological integrity and protect against neurodegeneration in prion diseases are still unclear. Here we report that after stimulation with the neurotoxic PrP106-126 fragment in primary cortical neurons, REST translocates from the cytoplasm to the nucleus and protects neurons from harmful effects of PrP106-126. Overexpression of REST reduces pathological damage and abnormal biochemical alterations of neurons induced by PrP106-126 and maintains neuronal viability by stabilizing the level of pro-survival protein FOXO1 and inhibiting the permeability of the mitochondrial outer membrane, release of cytochrome c from mitochondria to cytoplasm and the activation of Capase3. Conversely, knockdown of REST exacerbates morphological damage and inhibits the expression of FOXO1. Additionally, by overexpression or knockdown of LRP6, we further show that LRP6-mediated Wnt-ß-catenin signaling partly regulates the expression of REST. Collectively, we demonstrate for the first time novel neuroprotective function of REST in prion diseases and hypothesise that the LRP6-Wnt-ß-catenin/REST signaling plays critical and collaborative roles in neuroprotection. This signaling of neuronal survival regulation could be explored as a viable therapeutic target for prion diseases and associated neurodegenerative diseases.

Aging periosteal progenitor cells have reduced regenerative responsiveness to bone injury and to the anabolic actions of PTH 1-34 treatment.

A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7 days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. ß-Catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active ß-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. ß-Catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects.