Androgens up-regulate transcription of the Notch inhibitor Numb in C2C12 myoblasts via Wnt/ß-catenin signaling to T cell factor elements in the Numb promoter.

Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3ß. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which ß-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling.

The central nervous system (CNS)-independent anti-bone-resorptive activity of muscle contraction and the underlying molecular and cellular signatures.

BACKGROUND: Mechanisms by which muscle regulates bone are poorly understood. RESULTS: Electrically stimulated muscle contraction reversed elevations in bone resorption and increased Wnt signaling in bone-derived cells after spinal cord transection. CONCLUSION: Muscle contraction reduced resorption of unloaded bone independently of the CNS, through mechanical effects and, potentially, nonmechanical signals (e.g. myokines). SIGNIFICANCE: The study provides new insights regarding muscle-bone interactions. Muscle and bone work as a functional unit. Cellular and molecular mechanisms underlying effects of muscle activity on bone mass are largely unknown. Spinal cord injury (SCI) causes muscle paralysis and extensive sublesional bone loss and disrupts neural connections between the central nervous system (CNS) and bone. Muscle contraction elicited by electrical stimulation (ES) of nerves partially protects against SCI-related bone loss. Thus, application of ES after SCI provides an opportunity to study the effects of muscle activity on bone and roles of the CNS in this interaction, as well as the underlying mechanisms. Using a rat model of SCI, the effects on bone of ES-induced muscle contraction were characterized. The SCI-mediated increase in serum C-terminal telopeptide of type I collagen (CTX) was completely reversed by ES. In ex vivo bone marrow cell cultures, SCI increased the number of osteoclasts and their expression of mRNA for several osteoclast differentiation markers, whereas ES significantly reduced these changes; SCI decreased osteoblast numbers, but increased expression in these cells of receptor activator of NF-κB ligand (RANKL) mRNA, whereas ES increased expression of osteoprotegerin (OPG) and the OPG/RANKL ratio. A microarray analysis revealed that ES partially reversed SCI-induced alterations in expression of genes involved in signaling through Wnt, FSH, parathyroid hormone (PTH), oxytocin, and calcineurin/nuclear factor of activated T-cells (NFAT) pathways. ES mitigated SCI-mediated increases in mRNA levels for the Wnt inhibitors DKK1, sFRP2, and sclerostin in ex vivo cultured osteoblasts. Our results demonstrate an anti-bone-resorptive activity of muscle contraction by ES that develops rapidly and is independent of the CNS. The pathways involved, particularly Wnt signaling, suggest future strategies to minimize bone loss after immobilization.

Electrical stimulation modulates Wnt signaling and regulates genes for the motor endplate and calcium binding in muscle of rats with spinal cord transection.

BACKGROUND: Spinal cord injury (SCI) results in muscle atrophy and a shift of slow oxidative to fast glycolytic fibers. Electrical stimulation (ES) at least partially restores muscle mass and fiber type distribution. The objective of this study was to was to characterize the early molecular adaptations that occur in rat soleus muscle after initiating isometric resistance exercise by ES for one hour per day for 1, 3 or 7 days when ES was begun 16 weeks after SCI. Additionally, changes in mRNA levels after ES were compared with those induced in soleus at the same time points after gastrocnemius tenotomy (GA). RESULTS: ES increased expression of Hey1 and Pitx2 suggesting increased Notch and Wnt signaling, respectively, but did not normalize RCAN1.4, a measure of calcineurin/NFAT signaling, or PGC-1ß mRNA levels. ES increased PGC-1α expression but not that of slow myofibrillar genes. Microarray analysis showed that after ES, genes coding for calcium binding proteins and nicotinic acetylcholine receptors were increased, and the expression of genes involved in blood vessel formation and morphogenesis was altered. Of the 165 genes altered by ES only 16 were also differentially expressed after GA, of which 12 were altered in the same direction by ES and GA. In contrast to ES, GA induced expression of genes related to oxidative phosphorylation. CONCLUSIONS: Notch and Wnt signaling may be involved in ES-induced increases in the mass of paralyzed muscle. Molecular adaptations of paralyzed soleus to resistance exercise are delayed or defective compared to normally innervated muscle.

Evaluation of a Telehealth-Based Pharmacist Led Chronic Care Management Program.

Background: Clinical pharmacy services improve several patient chronic disease outcomes. This review evaluates a pharmacist-led chronic care management (CCM) program partnered with a health system for patient outcomes and sustainability. Methods: A mixed methods evaluation based on the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework was completed. Patient A1c and blood pressure readings were retrospectively collected from the electronic health record from August 2018-April 2022. Patients that completed >4 CCM visits with a diagnosis of diabetes and/or hypertension were included. Results: 557 patients enrolled, 53 had uncontrolled systolic blood pressure (SBP), SBP >130 mmHg. Average SBP at baseline was 141.0 mmHg and average SBP at 6 months was 130.2 mmHg, (P < .001). 76 patients had uncontrolled diabetes, A1c > 7%. Average A1c at baseline = 9.1% and average A1c at 6 months = 8.3%, (P < .001). 4464 CCM visits with 247 disease-state targeted patients were completed over 44-month with a 100% adoption rate across clinic locations. Implementation facilitators included patient medication cost concerns, disease burden, provider revenue generation, CCM dedicated software, streamlined call process, and remote EMR access. Implementation barriers included provider discomfort "selling the program," potential patient costs, unclear need from patient, pharmacists not considered providers, pharmacist cost, multi-platform software, reprioritized stakeholder support, and lack of partner site diversification. Program maintenance showed revenue generation was $5925.31-$8879.89 from August 2021-May 2022 and profitability was $3385.61-$1614.23. Conclusion: This study provides lessons learned, strategies for implementation, and ideas for process efficiencies leading to maintenance of a telehealth pharmacist-led CCM service.

Randomized peptide assemblies for enhancing immune responses to nanomaterials.

Biomaterials capable of inducing immune responses with minimal associated inflammation are of interest in applications ranging from tissue repair to vaccines. Here we report the design of self-assembling randomized polypeptide nanomaterials inspired by glatiramoids, an immunomodulatory class of linear random copolymers. We hypothesized that peptide self-assemblies bearing similar randomized polypeptides would similarly raise responses skewed toward Type 2 immunity and TH2 T-cell responses, additionally strengthening responses to co-assembled peptide epitopes in the absence of adjuvant. We developed a method for synthesizing self-assembling peptides terminated with libraries of randomized polypeptides (termed KEYA) with good batch-to-batch reproducibility. These peptides formed regular nanofibers and raised strong antibody responses without adjuvants. KEYA modifications dramatically improved uptake of peptide nanofibers in vitro by antigen presenting cells, and served as strong B-cell and T-cell epitopes in vivo, enhancing immune responses against epitopes relevant to influenza and chronic inflammation while inducing a KEYA-specific Type 2/TH2/IL-4 phenotype. KEYA modifications also increased IL-4 production by T cells, extended the residence time of nanofibers, induced no measurable swelling in footpad injections, and decreased overall T cell expansion compared to unmodified nanofibers, further suggesting a TH2 T-cell response with minimal inflammation. Collectively, this work introduces a biomaterial capable of raising strong Type 2/TH2/IL-4 immune responses, with potential applications ranging from vaccination to tissue repair.

Dose-related differences in the regional pattern of cannabinoid receptor adaptation and in vivo tolerance development to delta9-tetrahydrocannabinol.

Chronic treatment with Delta(9)-tetrahydrocannabinol (THC) produces tolerance to cannabinoid-mediated behaviors and region-specific adaptation of brain cannabinoid receptors. However, the relationship between receptor adaptation and tolerance is not well understood, and the dose-response relationship of THC-induced cannabinoid receptor adaptation is unknown. This study assessed cannabinoid receptor function in the brain and cannabinoid-mediated behaviors after chronic treatment with different dosing regimens of THC. Mice were treated twice per day for 6.5 days with the following: vehicle, 10 mg/kg THC, or escalating doses of 10 to 20 to 30 or 10 to 30 to 60 mg/kg THC. Tolerance to cannabinoid-mediated locomotor inhibition, ring immobility, antinociception, and hypothermia was produced by both ramping THC-dose paradigms. Administration of 10 mg/kg THC produced less tolerance development, the magnitude of which depended upon the particular behavior. Decreases in cannabinoid-mediated G-protein activation, which varied with treatment dose and region, were observed in autoradiographic and membrane guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS)-binding assays in brains from THC-treated mice. Agonist-stimulated [(35)S]GTPgammaS binding was reduced in the hippocampus, cingulate cortex, periaqueductal gray, and cerebellum after all treatments. Decreased agonist-stimulated [(35)S]GTPgammaS binding in the caudate-putamen, nucleus accumbens, and preoptic area occurred only after administration of 10 to 30 to 60 mg/kg THC, and no change was found in the globus pallidus or entopeduncular nucleus after any treatment. Changes in the CB(1) receptor B(max) values also varied by region, with hippocampus and cerebellum showing reductions after all treatments and striatum/globus pallidus showing effects only at higher dosing regimens. These results reveal that tolerance and CB(1) receptor adaptation exhibit similar dose-dependent development, and they are consistent with previous studies demonstrating less cannabinoid receptor adaptation in striatal circuits.

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns.

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting-the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increase in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA ("spatial noise") that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. These results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.

A Soluble Activin Receptor IIB Fails to Prevent Muscle Atrophy in a Mouse Model of Spinal Cord Injury.

Myostatin (MST) is a potent regulator of muscle growth and size. Spinal cord injury (SCI) results in marked atrophy of muscle below the level of injury. Currently, there is no effective pharmaceutical treatment available to prevent sublesional muscle atrophy post-SCI. To determine whether inhibition of MST with a soluble activin IIB receptor (RAP-031) prevents sublesional SCI-induced muscle atrophy, mice were randomly assigned to the following groups: Sham-SCI; SCI+Vehicle group (SCI-VEH); and SCI+RAP-031 (SCI-RAP-031). SCI was induced by complete transection at thoracic level 10. Animals were euthanized at 56 days post-surgery. RAP-031 reduced, but did not prevent, body weight loss post-SCI. RAP-031 increased total lean tissue mass compared to SCI-VEH (14.8%). RAP-031 increased forelimb muscle mass post-SCI by 38% and 19% for biceps and triceps, respectively (p < 0.001). There were no differences in hindlimb muscle weights between the RAP-031 and SCI-VEH groups. In the gastrocnemius, messenger RNA (mRNA) expression was elevated for interleukin (IL)-6 (8-fold), IL-1ß (3-fold), and tumor necrosis factor alpha (8-fold) in the SCI-VEH, compared to the Sham group. Muscle RING finger protein 1 mRNA was 2-fold greater in the RAP-031 group, compared to Sham-SCI. RAP-031 did not influence cytokine expression. Bone mineral density of the distal femur and proximal tibia were decreased post-SCI (-26% and -28%, respectively) and were not altered by RAP-031. In conclusion, MST inhibition increased supralesional muscle mass, but did not prevent sublesional muscle or bone loss, or the inflammation in paralyzed muscle.

Toluene inhalation produces a conditioned place preference in rats.

Toluene is a widely abused solvent with demonstrated addictive potential in humans. Here we explore if conditioned place preference can be used to study the abuse-related effects of inhaled toluene in rats. Animals were confined to a distinctive compartment of a three-compartment chamber while exposed to toluene vapor and later tested for preference for that compartment compared to appropriate control subjects. In this study, a flame ionization detector was used for on-line monitoring of toluene vapor concentrations inside the conditioning apparatus coupled with computerized recording of the time spent by the animals on the test day in each of the chambers. Sprague-Dawley rats were exposed to 810, 1895 or 4950 ppm of toluene vapors in either the black or white compartment during 30-min pairing sessions given every other day alternating with air exposure for the total of six pairings for each treatment. Rats that received air in both sides (control group) did not show any preference for either side with approximately equal time spent in each compartment on the test day (241 +/- 33 and 234 +/- 34 s, for white and black box, respectively). However, the 1895- and 4950-ppm test groups, but not the 810-ppm group, demonstrated a significant preference for the side paired with toluene exposure. When a subsequent test session was performed during toluene exposures, no conditioned place preference was observed. Thus, toluene produced a clear conditioned place preference that appears to be most evident when animals are not intoxicated. This procedure should be useful for further studies of the abuse-related effects of abused inhalants.

Alterations of serotonin 2C and 2A receptors in response to T10 spinal cord transection in rats.

Recent studies reported that a recovery of motoneurons after spinal cord transection at the sacrocaudal level may depend on adaptive alterations of the serotonin 2C (5-HT(2C)R) and 2A (5-HT(2C)R) receptor function via changes in mRNA editing or protein expression, respectively. It has been suggested that depletion of serotonergic input may drive these adaptations. Here, mRNA editing and/or expression of 5-HT(2C)R and 5-HT(2A)R was evaluated in rats that sustained a complete transection at the thoracic (T10) level. While 5-HT(2A)R mRNA expression was upregulated below the site of spinal cord injury (SCI), no changes in 5-HT(2C)R mRNA editing or expression were detected. These findings argue against the hypothesis that 5-HT(2C)R editing is regulated by extracellular serotonin levels. Rather, it appears that the editing process is just one of the ways in which excitability of motor neurons can be restored following SCI. To this end, the influence of excitatory locomotor circuits on motor neurons in the thoracic spinal cord of rats requires further exploration.

Nandrolone, an anabolic steroid, stabilizes Numb protein through inhibition of mdm2 in C2C12 myoblasts.

Nandrolone, an anabolic steroid, slows denervation atrophy of rat muscle, prevents denervation-induced nuclear accumulation of intracellular domain of the Notch receptor, and elevates expression of Numb. Numb acts as an inhibitor of Notch signaling and promotes myogenic differentiation of satellite cells. Turnover of Numb is regulated by mdm2, an E3 ubiquitin ligase. With these considerations in mind, we investigated the effects of nandrolone on the expression of Numb and mdm2 proteins and determined the effect of mdm2 on nandrolone-induced alterations in Numb protein in C2C12 myoblasts. When C2C12 cells were cultured in a medium favoring differentiation (Dulbecco modified Eagle medium containing 2% horse serum), nandrolone up-regulated Numb protein levels in a time-dependent manner and prolonged Numb protein half-life from 10 to 18 hours. In contrast, nandrolone reduced the expression of mdm2 protein. To determine whether the decreased mdm2 expression induced by nandrolone was responsible for the increased levels and prolonged half-life of Numb protein in this cell line, mdm2-small interfering RNA (siRNA) was employed to inhibit mdm2 expression. Compared to cells transfected with scrambled siRNA (negative control), transfection with mdm2-siRNA increased basal Numb protein expression but abolished the further increase in Numb protein levels by nandrolone. In addition, transfection of mdm2-siRNA mimicked the effect of nandrolone to prolong the half-life of Numb protein. Moreover, when C2C12 cells were forced to overexpress mdm2, there was a significant decline in the expression of both basal and inducible Numb protein. Our data suggest that nandrolone, by a novel mechanism for this agent in a muscle cell type, increases Numb protein levels in C2C12 myoblasts by stabilizing Numb protein against degradation, at least in part, via suppression of mdm2 expression.

Synergistic transcriptional activation by one regulatory protein in response to two metabolites.

BenM is a LysR-type bacterial transcriptional regulator that controls aromatic compound degradation in Acinetobacter sp. ADP1. Here, in vitro transcription assays demonstrated that two metabolites of aromatic compound catabolism, benzoate and cis,cis-muconate, act synergistically to activate gene expression. The level of BenM-regulated benA transcription was significantly higher in response to both compounds than the combined levels due to each alone. These compounds also were more effective together than they were individually in altering the DNase I footprint patterns of BenM-DNA complexes. This type of dual-inducer synergy provides great potential for rapid and large modulations of gene expression and may represent an important, and possibly widespread, feature of transcriptional control.