Acute Phosphate Restriction Leads to Impaired Fracture Healing and Resistance to BMP-2

Hypophosphatemia leads to rickets and osteomalacia, the latter of which results in decreased biomechanical integrity of bones, accompanied by poor fracture healing. Impaired phosphate-dependent apoptosis of hypertrophic chondrocytes is the molecular basis for rickets. However, the underlying pathophysiology of impaired fracture healing has not been characterized previously. To address the role of phosphate in fracture repair, mice were placed on a phosphate-restricted diet 2 days prior to or 3 days after induction of a mid-diaphyseal femoral fracture to assess the effects of phosphate deficiency on the initial recruitment of mesenchymal stem cells and their subsequent differentiation. Histologic and micro-computed tomographic (µCT) analyses demonstrated that both phosphate restriction models dramatically impaired fracture healing primarily owing to a defect in differentiation along the chondrogenic lineage. Based on Sox9 and Sox5 mRNA levels, neither the initial recruitment of cells to the callus nor their lineage commitment was effected by hypophosphatemia. However, differentiation of these cells was impaired in association with impaired bone morphogenetic protein (BMP) signaling. In vivo ectopic bone-formation assays and in vitro investigations in ST2 stromal cells confirmed that phosphate restriction leads to BMP-2 resistance. Marrow ablation studies demonstrate that hypophosphatemia has different effects on injury-induced intramembranous bone formation compared with endochondral bone formation. Thus phosphate plays an important role in the skeleton that extends beyond mineralized matrix formation and growth plate maturation and is critical for endochondral bone repair. © 2010 American Society for Bone and Mineral Research

A Prescription Digital Therapeutic to Support Unsupervised Buprenorphine Initiation for Patients With Opioid Use Disorder: Protocol for a Proof-of-Concept Study

BackgroundHome-based (unsupervised) buprenorphine initiation is considered safe and effective, yet many patients report barriers to successful treatment initiation. Prescription digital therapeutics (PDTs) are software-based disease treatments regulated by the US Food and Drug Administration (FDA). The reSET-O PDT was authorized by the FDA in 2018 and delivers behavioral treatment for individuals receiving buprenorphine for opioid use disorder (OUD). A prototype PDT (PEAR-002b) designed for use with reSET-O was developed to assist in unsupervised buprenorphine initiation. ObjectiveThe primary objective of this pilot study is to evaluate the acceptability of PEAR-002b in individuals with OUD who use it to support buprenorphine initiation, their unsupervised buprenorphine initiation success rate, and their medication adherence. MethodsTen adults with OUD will be recruited for acceptability and feasibility testing. Outcomes will be assessed using week-1 visit attendance, participant interviews and satisfaction surveys, and urine drug screening (UDS). Three tools will be used in the study: PEAR-002b, reSET-O, and EmbracePlus. PEAR-002b includes a new set of features designed for use with reSET-O. The mechanism of action for the combined PEAR-002b and reSET-O treatment is a program of medication dosing support during week 1 of the initiation phase, cognitive behavioral therapy, and contingency management. During the medication initiation phase, participants are guided through a process to support proper medication use. PEAR-002b advises them when to take their buprenorphine based on provider inputs (eg, starting dose), self-reported substance use, and self-reported withdrawal symptoms. This study also administers the EmbracePlus device, a medical-grade smartwatch, to pilot methods for collecting physiologic data (eg, heart rate and skin conductance) and evaluate the device’s potential for use along with PDTs that are designed to improve OUD treatment initiation. Home buprenorphine initiation success will be summarized as the proportion of participants attending the post–buprenorphine initiation visit (week 1) and the proportion of participants who experience buprenorphine initiation–related adverse events (eg, precipitated withdrawal). Acceptability of PEAR-002b will be evaluated based on individual participants’ ratings of ease of use, satisfaction, perceived helpfulness, and likelihood of recommending PEAR-002b. Medication adherence will be evaluated by participant self-report data and confirmed by UDS. UDS data will be summarized as the mean of individual participants’ proportion of total urine samples testing positive for buprenorphine or norbuprenorphine over the 4-week study. ResultsThis project was funded in September 2019. As of September 2022, participant enrollment is ongoing. ConclusionsThis is the first study to our knowledge to develop a PDT that assists with unsupervised buprenorphine initiation with the intent to better support patients and prescribers during this early phase of treatment. This pilot study will assess the acceptability and utility of a digital therapeutic to assist individuals with OUD with unsupervised buprenorphine initiation. Trial RegistrationClinicalTrials.gov NCT05412966; https://clinicaltrials.gov/ct2/show/NCT05412966 International Registered Report Identifier (IRRID)PRR1-10.2196/4312

The Vitamin D Receptor Is Required for Activation of cWnt and Hedgehog Signaling in Keratinocytes

Alopecia (hair loss) in vitamin D receptor (VDR)-null mice is due to absence of ligand-independent actions of the VDR that are required for initiation of postmorphogenic hair cycles. Investigations were undertaken to determine whether the VDR is required for the induction of signaling pathways that play an important role in this process. The induction of cWnt and hedgehog target genes that characterizes early anagen was found to be dramatically attenuated in VDR(−/−) mice, relative to wild-type (WT) mice. To determine whether this reflects impaired responsiveness to cWnt ligands, in vitro studies were performed in primary keratinocytes. These studies demonstrated impaired induction of cWnt target genes in response to Wnt3a in VDR(−/−) keratinocytes, relative to wild-type keratinocytes. Chromatin immunoprecipitation analyses revealed that the VDR was recruited to the regulatory regions of cWnt and hedgehog target genes in WT keratinocytes but not in VDR(−/−) or Lef1(−/−) keratinocytes. Lef1 was enriched on these same regulatory regions in WT keratinocytes but not in VDR(−/−) keratinocytes. In vivo studies were performed to determine whether activation of the hedgehog pathway could bypass the defect in cWnt signaling observed in the absence of the unliganded VDR. In WT, but not VDR(−/−), mice, hedgehog agonist treatment resulted in an induction of cWnt and hedgehog target genes and the generation of mature anagen hair follicles. Thus, these studies demonstrate that the unliganded VDR interacts with regulatory regions in the cWnt and hedgehog target genes and is required for the induction of these pathways during the postnatal hair cycle

Lymphoid Enhancer-binding Factor-1 (LEF1) Interacts with the DNA-binding Domain of the Vitamin D Receptor*

Ligand-independent actions of the vitamin D receptor (VDR) are required for normal post-morphogenic hair cycles; however, the molecular mechanisms by which the VDR exerts these actions are not clear. Previous studies demonstrated impaired regulation of the canonical Wnt signaling pathway in primary keratinocytes lacking the VDR. To identify the key effector of canonical Wnt signaling that interacts with the VDR, GST pulldown studies were performed. A novel interaction between the VDR and LEF1 (lymphoid enhancer-binding factor-1) that is independent of β-catenin was identified. This interaction is dependent upon sequences within the N-terminal region of the VDR, a domain required for VDR-DNA interactions and normal hair cycling in mice. Mutation of specific residues within the N-terminal region of the VDR not only abrogated interactions between the VDR and LEF1 but also impaired the ability of the VDR to enhance Wnt signaling in vdr−/− primary keratinocytes. Thus, this study demonstrates a novel interaction between the VDR and LEF1 that is mediated by the DNA-binding domain of the VDR and that is required for normal canonical Wnt signaling in keratinocytes

Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. The precise mode of action and the full spectrum of activities of the vitamin D hormone, 1,25-dihydroxyvitamin D [1,25-(OH)2D], can now be better evaluated by critical analysis of mice with engineered deletion of the vitamin D receptor (VDR). Absence of a functional VDR or the key activating enzyme, 25-OHD-1α-hydroxylase (CYP27B1), in mice creates a bone and growth plate phenotype that mimics humans with the same congenital disease or severe vitamin D deficiency. The intestine is the key target for the VDR because high calcium intake, or selective VDR rescue in the intestine, restores a normal bone and growth plate phenotype